Compositions and methods

ABSTRACT

The present invention is directed to a polypeptide which comprises: (i) an Rv2386c protein sequence; (ii) a variant of an Rv2386c protein sequence; of (iii) an immunogenic fragment of an Rv2386c protein sequence. In other aspects the invention is directed to associated polynucleotides, fusion proteins and methods for the treatment or prevention of  tuberculosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/929,232, nowabandoned, which is a continuation of U.S. Ser. No. 13/055,787, nowabandoned, which is the US National Stage of International ApplicationNo. PCT/EP2009/059585, filed 24 Jul. 2009, which claims benefit of thefiling date of U.S. Provisional Application No. 61/083,699, filed 25Jul. 2008, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to polypeptides and polynucleotides foruse in the treatment or prevention of tuberculosis, in particular foruse in the treatment or prevention of latent tuberculosis and in theprevention or delay of reactivation of tuberculosis (and also to relatedmethods). The present invention further relates to pharmaceutical andimmunogenic compositions comprising said polypeptides andpolynucleotides, and to methods for the diagnosis of tuberculosis (inparticular latent tuberculosis).

BACKGROUND OF THE INVENTION

Tuberculosis (TB) is a chronic infectious disease caused by infectionwith Mycobacterium tuberculosis and other Mycobacterium species. It is amajor disease in developing countries, as well as an increasing problemin developed areas of the world. More than 2 billion people are believedto be infected with TB bacilli, with about 9.2 million new cases of TBand 1.7 million deaths each year. 10% of those infected with TB bacilliwill develop active TB, each person with active TB infecting an averageof 10 to 15 others per year. While annual incidence rates have peakedglobally, the number of deaths and cases is still rising due topopulation growth (World Health Organisation Tuberculosis Facts 2008).

Mycobacterium tuberculosis infects individuals through the respiratoryroute. Alveolar macrophages engulf the bacterium, but it is able tosurvive and proliferate by inhibiting phagosome fusion with acidiclysosomes. A complex immune response involving CD4+ and CD8+ T cellsensues, ultimately resulting in the formation of a granuloma. Central tothe success of Mycobacterium tuberculosis as a pathogen is the fact thatthe isolated, but not eradicated, bacterium may persist for longperiods, leaving an individual vulnerable to the later development ofactive TB.

Fewer than 5% of infected individuals develop active TB in the firstyears after infection. The granuloma can persist for decades and isbelieved to contain live Mycobacterium tuberculosis in a state ofdormancy, deprived of oxygen and nutrients. However, recently it hasbeen suggested that the majority of the bacteria in the dormancy stateare located in non-macrophage cell types spread throughout the body(Locht et al, Expert Opin. Biol. Ther. 2007 7(11):1665-1677). Thedevelopment of active TB occurs when the balance between the host'snatural immunity and the pathogen changes, for example as a result of animmunosuppressive event (Anderson P Trends in Microbiology 200715(1):7-13; Ehlers S Infection 2009 37(2):87-95).

A dynamic hypothesis describing the balance between latent TB and activeTB has also been proposed (Cardana P-J Inflammation & Allergy—DrugTargets 2006 6:27-39; Cardana P-J Infection 2009 37(2):80-86).

Although an infection may be asymptomatic for a considerable period oftime, the active disease is most commonly manifested as an acuteinflammation of the lungs, resulting in tiredness, weight loss, feverand a persistent cough. If untreated, serious complications and deathtypically result.

Tuberculosis can generally be controlled using extended antibiotictherapy, although such treatment is not sufficient to prevent the spreadof the disease. Infected individuals may be asymptomatic, butcontagious, for some time. In addition, although compliance with thetreatment regimen is critical, patient behaviour is difficult tomonitor. Some patients do not complete the course of treatment, whichcan lead to ineffective treatment and the development of drugresistance.

Multidrug-resistant TB (MDR-TB) is a form which fails to respond tofirst line medications. 5% of all TB cases are MDR-TB, with an estimated490,000 new MDR-TB cases occurring each year. Extensively drug-resistantTB (XDR-TB) occurs when resistance to second line medications developson top of MDR-TB. It is estimated that 40,000 new cases of the virtuallyuntreatable XDR-TB arise annually (World Health OrganisationTuberculosis Facts 2008).

Even if a full course of antibiotic treatment is completed, infectionwith M. tuberculosis may not be eradicated from the infected individualand may remain as a latent infection that can be reactivated.

In order to control the spread of tuberculosis, effective vaccinationand accurate early diagnosis of the disease are of utmost importance.

Diagnosis of latent TB infection is commonly achieved using thetuberculin skin test, which involves intradermal exposure to tuberculinprotein-purified derivative (PPD). Antigen-specific T cell responsesresult in measurable induration at the injection site by 48-72 hoursafter injection, which indicates exposure to mycobacterial antigens.Sensitivity and specificity have, however, been a problem with thistest, and individuals vaccinated with BCG cannot always be easilydistinguished from infected individuals (this is particularly importantin light of the fact that BCG does not protect against latentinfection). In general, individuals who have received BCG but are notinfected by M. tuberculosis show a PPD reaction below 10 mm in diameterwhereas people who have a PPD reaction above 10 mm in diameter areconsidered to have been infected by M. tuberculosis. However, this ruleis not applicable to individuals with immunosuppression due to HIVinfection, which may result in a PPD reaction below 10 mm in diameter);or in endemic countries, where people infected by non-tuberculosismycobacteria can show a PPD reaction above 10 mm in diameter.

Progress over recent years has seen the development of in vitro T cellbased assays, based on interferon-gamma release and using antigens whichare more specific to M. tuberculosis than PPD, namely ESAT-6 and CFP-10.These high specificity tests appear to be at least as sensitive as thetuberculin skin test and also demonstrate less cross-reactivity due toBCG vaccination. See Pai M et al Expert Rev. Mol. Diagn. 20066(3):413-422 for a recent review of latent TB diagnosis. However, sinceESAT-6/CFP-10 are early stage antigens, assays based on ESAT-6/CFP-10may only perform optimally in recently infected people. Consequently,the identification of new antigens specifically associated with latenttuberculosis may aid the development of more sensitive assays that coulddetect longer-term latent infections.

There remains a need for effective strategies for the treatment andprevention of tuberculosis, in particular the treatment and preventionof latent TB and the prevention of reactivation of TB.

BRIEF SUMMARY OF THE INVENTION

The present invention relates generally to the identification of Rv2386cas a TB antigen (in particular an antigen associated with latent TB) andto related methods and uses in the prevention and treatment of TB,especially the prevention and treatment of latent TB and the preventionor delay of TB reactivation.

The present invention provides an isolated polypeptide which comprises:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence.

The present invention also provides a polypeptide which comprises:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence;        for use as a medicament.

A further aspect of the invention relates to a method for the treatmentor prevention of TB comprising the administration of a safe andeffective amount of a polypeptide comprising:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence;        to a subject in need thereof, wherein said polypeptide induces        an immune response, in particular an immune response against        Mycobacterium tuberculosis.

The use of a polypeptide comprising:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence;        in the manufacture of a medicament for the treatment or        prevention of TB, represents another aspect of the invention.

The present invention provides an isolated polynucleotide comprising anucleic acid sequence encoding a polypeptide which comprises:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence.

Also provided is a polynucleotide comprising a nucleic acid sequenceencoding a polypeptide which comprises:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence;        for use as a medicament.

A further aspect of the invention relates to a method for the treatmentor prevention of TB comprising the administration of a safe andeffective amount of a polynucleotide comprising a nucleic acid sequenceencoding a polypeptide comprising:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence;        to a subject in need thereof, wherein said polynucleotide        induces an immune response, in particular an immune response        against Mycobacterium tuberculosis.

Use of a polynucleotide comprising a nucleic acid sequence encoding apolypeptide comprising:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence;        in the manufacture of a medicament for the treatment or        prevention of TB, represents another aspect of the invention.

Additionally, there is provided a pharmaceutical composition comprising:

-   -   (a) a polypeptide which comprises:        -   (i) an Rv2386c protein sequence;        -   (ii) a variant of an Rv2386c protein sequence; or        -   (iii) an immunogenic fragment of an Rv2386c protein            sequence; or    -   (b) a polynucleotide comprising a nucleic acid sequence encoding        the polypeptide of (a);    -   and    -   (c) a pharmaceutically acceptable carrier or excipient.

Further, there is provided an immunogenic composition comprising:

-   -   (a) a polypeptide which comprises:        -   (i) an Rv2386c protein sequence;        -   (ii) a variant of an Rv2386c protein sequence; or        -   (iii) an immunogenic fragment of an Rv2386c protein            sequence; or    -   (b) a polynucleotide comprising a nucleic acid sequence encoding        the polypeptide of (a);    -   and    -   (c) a non-specific immune response enhancer.

Suitably the pharmaceutical and immunogenic composition comprise acombination of an Rv2386c and a second heterologous tuberculosis antigencomponent.

Also provided is an expression vector comprising a nucleic acid sequenceencoding a polypeptide which comprises:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence.

Host cells, transformed with said expression vector, form a furtheraspect of the invention. Additionally provided is a host cell whichrecombinantly expresses a polypeptide which comprises:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence.

Further, there is provided a method for the production of a polypeptidecomprising:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence;        said method comprising the step of recombinantly expressing said        polypeptide within a host cell.

Additionally provided is an antibody or fragment thereof whichspecifically binds to a polypeptide comprising:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence.

The use of said antibodies in diagnosis is also provided (such asmethods for the diagnosis of tuberculosis comprising determining thepresence of an antibody or fragment thereof which specifically binds tothe polypeptides of the invention in a biological sample from a testsubject).

Also provided are diagnostic kits comprising:

-   -   (a) a polypeptide of the present invention;    -   (b) apparatus sufficient to contact said polypeptide with a        sample (e.g. whole blood or more suitably PBMC) from an        individual; and    -   (c) means to quantify the T cell response of the sample.

Another aspect of the invention relates to a diagnostic kit comprising:

-   -   (a) a polypeptide of the present invention; and    -   (b) apparatus sufficient to contact said polypeptide with the        dermal cells of a patient.

In one embodiment the subject receiving a polypeptide, polynucleotide orcomposition of the invention may have active tuberculosis (e.g. activeinfection by M. tuberculosis). In a second embodiment the subject mayhave latent tuberculosis (e.g. dormant infection by M. tuberculosis). Ina third embodiment the subject may be free from tuberculosis (e.g. freefrom infection by M. tuberculosis).

A subject receiving a polypeptide, polynucleotide or composition of theinvention may have previously been vaccinated for tuberculosis (e.g.vaccinated against infection by M. tuberculosis), such as having beenvaccinated with Bacillus Calmette-Guerin (BCG). Alternatively, a subjectreceiving a polypeptide, polynucleotide or composition of the inventionmay not have previously been vaccinated for tuberculosis (e.g. notvaccinated against infection by M. tuberculosis), such as not havingbeen vaccinated with Bacillus Calmette-Guerin (BCG).

DESCRIPTION OF THE FIGURES

FIG. 1: Percentage of CD4 and CD8 cells from immunised CB6F1 miceexpressing IFN-gamma and/or IL-2 and/or TNF-alpha cytokines at day 21(i.e. 7 days post second immunisation).

FIG. 2: Cytokine profile at day 21 (i.e. 7 days post secondimmunisation) of the antigen specific CD4 response in immunised CB6F1mice.

FIG. 3: Cytokine profile at day 21 (i.e. 7 days post secondimmunisation) of the antigen specific CD8 response in immunised CB6F1mice.

FIG. 4: Percentage of CD4 and CD8 cells from immunised CB6F1 miceexpressing IFN-gamma and/or IL-2 and/or TNF-alpha cytokines at day 35(i.e. 7 days post third immunisation).

FIG. 5: Cytokine profile at day 35 (i.e. 7 days post third immunisation)of the antigen specific CD4 response in immunised CB6F1 mice.

FIG. 6: Cytokine profile at day 35 (i.e. 7 days post third immunisation)of the antigen specific CD8 response in immunised CB6F1 mice.

FIG. 7: Percentage of CD4 and CD8 cells from immunised C57BL/6 miceexpressing IFN-gamma and/or IL-2 and/or TNF-alpha cytokines at day 21(i.e. 7 days post second immunisation).

FIG. 8: Cytokine profile at day 21 (i.e. 7 days post secondimmunisation) of the antigen specific CD4 response in immunised C57BL/6mice.

FIG. 9: Cytokine profile at day 21 (i.e. 7 days post secondimmunisation) of the antigen specific CD8 response in immunised C57BL/6mice.

DESCRIPTION OF THE LISTED SEQUENCES

-   -   SEQ ID No: 1: polypeptide sequence of Rv2386c from M.        tuberculosis H37Rv strain.    -   SEQ ID No: 2: polynucleotide sequence of Rv2386c from M.        tuberculosis H37Rv strain.    -   SEQ ID No: 3: polypeptide sequence of Rv2386c from M.        tuberculosis CDC1551 strain.    -   SEQ ID No: 4: polypeptide sequence of Rv2386c from M.        tuberculosis F11 strain.    -   SEQ ID No: 5: polypeptide sequence of Rv2386c from M.        tuberculosis Haarlem A strain.    -   SEQ ID No: 6: polypeptide sequence of Rv2386c from M.        tuberculosis C strain.    -   SEQ ID No: 7: polypeptide sequence of Rv2386c from BCG.    -   SEQ ID No: 8: polypeptide sequence of Mtb8.4.    -   SEQ ID No: 9: polypeptide sequence of Mtb9.8.    -   SEQ ID No: 10: polypeptide sequence of Mtb9.9.    -   SEQ ID No: 11: polypeptide sequence of Ra12.    -   SEQ ID No: 12: polypeptide sequence of Ra35.    -   SEQ ID No: 13: polypeptide sequence of TbH9.    -   SEQ ID No: 14: polypeptide sequence of Mtb40.    -   SEQ ID No: 15: polypeptide sequence of Mtb41.    -   SEQ ID No: 16: polypeptide sequence of ESAT-6.    -   SEQ ID No: 17: polypeptide sequence of Ag85A.    -   SEQ ID No: 18: polypeptide sequence of Ag85B.    -   SEQ ID No: 19: polypeptide sequence of alpha-crystallin.    -   SEQ ID No: 20: polypeptide sequence of MPT64.    -   SEQ ID No: 21: polypeptide sequence of Mtb32A.    -   SEQ ID No: 22: polypeptide sequence of Ser/Ala mutated mature        Mtb32A.    -   SEQ ID No: 23: polypeptide sequence of TB10.4.    -   SEQ ID No: 24: polypeptide sequence of Mtb72f.    -   SEQ ID No: 25: polypeptide sequence of M72.    -   SEQ ID No: 26: polypeptide sequence of Mtb71f.    -   SEQ ID No: 27: polypeptide sequence of M92 fusion.    -   SEQ ID No: 28: polypeptide sequence of M103 fusion.    -   SEQ ID No: 29: polypeptide sequence of M114 fusion.    -   SEQ ID No: 30: putative human CD4 cell epitope 1.    -   SEQ ID No: 31: putative human CD4 cell epitope 2.    -   SEQ ID No: 32: putative human CD4 cell epitope 3.    -   SEQ ID No: 33: putative human CD4 cell epitope 4.    -   SEQ ID No: 34: putative human CD4 cell epitope 5.    -   SEQ ID No: 35: putative human CD4 cell epitope 6.    -   SEQ ID No: 36: putative human CD4 cell epitope 7.    -   SEQ ID No: 37: putative human CD4 cell epitope 8.    -   SEQ ID No: 38: putative human CD4 cell epitope 9.    -   SEQ ID No: 39: putative human CD4 cell epitope 10.    -   SEQ ID No: 40: putative human CD4 cell epitope 11.    -   SEQ ID No: 41: putative human CD4 cell epitope 12.    -   SEQ ID No: 42: putative human CD4 cell epitope 13.    -   SEQ ID No: 43: putative human CD4 cell epitope 14.    -   SEQ ID No: 44: putative human CD4 cell epitope 15.    -   SEQ ID No: 45: putative human CD4 cell epitope 16.    -   SEQ ID No: 46: putative human CD4 cell epitope 17.    -   SEQ ID No: 47: putative human CD4 cell epitope 18.    -   SEQ ID No: 48: putative human CD4 cell epitope 19.    -   SEQ ID No: 49: putative human CD4 cell epitope 20.    -   SEQ ID No: 50: putative human CD4 cell epitope 21.    -   SEQ ID No: 51: putative human CD4 cell epitope 22.    -   SEQ ID No: 52: putative human CD4 cell epitope 23.    -   SEQ ID No: 53: putative human CD8 cell epitope 1.    -   SEQ ID No: 54: putative human CD8 cell epitope 2.    -   SEQ ID No: 55: putative human CD8 cell epitope 3.    -   SEQ ID No: 56: putative human CD8 cell epitope 4.    -   SEQ ID No: 57: putative human CD8 cell epitope 5.    -   SEQ ID No: 58: putative human CD8 cell epitope 6.    -   SEQ ID No: 59: putative human CD8 cell epitope 7.    -   SEQ ID No: 60: putative human CD8 cell epitope 8.    -   SEQ ID No: 61: putative human CD8 cell epitope 9.    -   SEQ ID No: 62: putative human CD8 cell epitope 10.    -   SEQ ID No: 63: putative human CD8 cell epitope 11.    -   SEQ ID No: 64: putative human CD8 cell epitope 12.    -   SEQ ID No: 65: putative human CD8 cell epitope 13.    -   SEQ ID No: 66: putative human CD8 cell epitope 14.    -   SEQ ID No: 67: putative human CD8 cell epitope 15.    -   SEQ ID No: 68: putative human CD8 cell epitope 16.    -   SEQ ID No: 69: putative human CD8 cell epitope 17.    -   SEQ ID No: 70: putative human CD8 cell epitope 18.    -   SEQ ID No: 71: putative human CD8 cell epitope 19.    -   SEQ ID No: 72: putative human CD8 cell epitope 20.    -   SEQ ID No: 73: putative human CD8 cell epitope 21.    -   SEQ ID No: 74: putative human CD8 cell epitope 22.    -   SEQ ID No: 75: putative human CD8 cell epitope 23.    -   SEQ ID No: 76: putative human CD8 cell epitope 24.    -   SEQ ID No: 77: putative human CD8 cell epitope 25.    -   SEQ ID No: 78: putative human CD8 cell epitope 26.    -   SEQ ID No: 79: putative human CD8 cell epitope 27    -   SEQ ID No: 80: putative human CD8 cell epitope 28.    -   SEQ ID No: 81: putative human CD8 cell epitope 29.    -   SEQ ID No: 82: putative human CD8 cell epitope 30.    -   SEQ ID No: 83: putative human CD8 cell epitope 31.    -   SEQ ID No: 84: putative human CD8 cell epitope 32.    -   SEQ ID No: 85: putative human CD8 cell epitope 33.    -   SEQ ID No: 86: putative human CD8 cell epitope 34.    -   SEQ ID No: 87: putative human CD8 cell epitope 35.    -   SEQ ID No: 88: putative human CD8 cell epitope 36.    -   SEQ ID No: 89: putative human CD8 cell epitope 37.    -   SEQ ID No: 90: putative human CD8 cell epitope 38.    -   SEQ ID No: 91: putative human CD8 cell epitope 39.    -   SEQ ID No: 92: putative human CD8 cell epitope 40.    -   SEQ ID No: 93: putative human CD8 cell epitope 41.    -   SEQ ID No: 94: putative human CD8 cell epitope 42.    -   SEQ ID No: 95: putative human CD8 cell epitope 43.    -   SEQ ID No: 96: putative human CD8 cell epitope 44.    -   SEQ ID No: 97: putative human CD8 cell epitope 45.    -   SEQ ID No: 98: putative human CD8 cell epitope 46.    -   SEQ ID No: 99: putative human CD8 cell epitope 47.    -   SEQ ID No: 100: putative human CD8 cell epitope 48.    -   SEQ ID No: 101: putative human CD8 cell epitope 49.    -   SEQ ID No: 102: putative human CD8 cell epitope 50.    -   SEQ ID No: 103: putative human CD8 cell epitope 51.    -   SEQ ID No: 104: putative human CD8 cell epitope 52.    -   SEQ ID No: 105: putative human CD8 cell epitope 53.    -   SEQ ID No: 106: putative human CD8 cell epitope 54.    -   SEQ ID No: 107: putative human CD8 cell epitope 55.    -   SEQ ID No: 108: putative human CD8 cell epitope 56.    -   SEQ ID No: 109: putative human CD8 cell epitope 57.    -   SEQ ID No: 110: putative human CD8 cell epitope 58.    -   SEQ ID No: 111: putative human CD8 cell epitope 59.    -   SEQ ID No: 112: putative human CD8 cell epitope 60.    -   SEQ ID No: 113: putative human CD8 cell epitope 61.    -   SEQ ID No: 114: putative human CD8 cell epitope 62.    -   SEQ ID No: 115: putative human CD8 cell epitope 63.    -   SEQ ID No: 116: putative human CD8 cell epitope 64.    -   SEQ ID No: 117: putative human CD8 cell epitope 65.    -   SEQ ID No: 118: putative human CD8 cell epitope 66.    -   SEQ ID No: 119: putative human CD8 cell epitope 67.    -   SEQ ID No: 120: putative human CD8 cell epitope 68.    -   SEQ ID No: 121: putative human CD8 cell epitope 69.    -   SEQ ID No: 122: putative human CD8 cell epitope 70.    -   SEQ ID No: 123: putative human CD8 cell epitope 71.    -   SEQ ID No: 124: putative human CD8 cell epitope 72.    -   SEQ ID No: 125: putative human CD8 cell epitope 73.    -   SEQ ID No: 126: putative human CD8 cell epitope 74.    -   SEQ ID No: 127: putative human CD8 cell epitope 75.    -   SEQ ID No: 128: putative human CD8 cell epitope 76.    -   SEQ ID No: 129: putative human CD8 cell epitope 77.    -   SEQ ID No: 130: putative human CD8 cell epitope 78.    -   SEQ ID No: 131: putative human CD8 cell epitope 79.    -   SEQ ID No: 132: putative human CD8 cell epitope 80.    -   SEQ ID No: 133: putative human CD8 cell epitope 81.    -   SEQ ID No: 134: putative human CD8 cell epitope 82.    -   SEQ ID No: 135: putative human CD8 cell epitope 83.    -   SEQ ID No: 136: putative human CD8 cell epitope 84.    -   SEQ ID No: 137: putative human CD8 cell epitope 85.    -   SEQ ID No: 138: putative human CD8 cell epitope 86.    -   SEQ ID No: 139: putative human CD8 cell epitope 87.    -   SEQ ID No: 140: putative human CD8 cell epitope 88.    -   SEQ ID No: 141: putative human CD8 cell epitope 89.    -   SEQ ID No: 142: putative human CD8 cell epitope 90.    -   SEQ ID No: 143: putative human CD8 cell epitope 91.    -   SEQ ID No: 144: putative human CD8 cell epitope 92.    -   SEQ ID No: 145: putative human CD8 cell epitope 93.    -   SEQ ID No: 146: putative human CD8 cell epitope 94.    -   SEQ ID No: 147: putative human CD8 cell epitope 95.    -   SEQ ID No: 148: putative human CD8 cell epitope 96.    -   SEQ ID No: 149: putative human CD8 cell epitope 97.    -   SEQ ID No: 150: putative human CD8 cell epitope 98.    -   SEQ ID No: 151: putative human CD8 cell epitope 99.    -   SEQ ID No: 152: putative human CD8 cell epitope 100.    -   SEQ ID No: 153: putative human CD8 cell epitope 101.    -   SEQ ID No: 154: putative human CD8 cell epitope 102.    -   SEQ ID No: 155: polypeptide sequence of Rv1753c from M.        tuberculosis H37Rv strain.    -   SEQ ID No: 156: polypeptide sequence of Rv2707c from M.        tuberculosis H37Rv strain.

DETAILED DESCRIPTION

Currently, vaccination with live bacteria is the most efficient methodfor inducing protective immunity. The most common Mycobacterium employedfor this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strainof M. bovis which was developed over 60 years ago. However, the safetyand efficacy of BCG is a source of controversy—while protecting againstsevere disease manifestation in children, BCG does not prevent theestablishment of latent TB or reactivation of pulmonary disease in adultlife. Additionally, some countries, such as the United States, do notvaccinate the general public with this agent.

Almost all new generation TB vaccines which are currently in clinicaldevelopment have been designed as pre-exposure vaccines. These includesubunit vaccines, which have been particularly effective in boostingimmunity induced by prior BCG vaccination, and advanced livemycobacterial vaccines which aim to replace BCG with more efficientand/or safer strains. Although these vaccines aim to improve resistanceto infection, they are likely to be less effective as post-exposure ortherapeutic vaccines in latent TB cases (Lin M Y et al Endocrine,Metabolic & Immune Disorders—Drug Targets 2008 8:15-29).

Several of the proteins which are strongly expressed during the earlystages of Mycobacterium infection have been shown to provide strongprotective efficacy in animal vaccination models. However, vaccinationwith antigens which are highly expressed during the early stages ofinfection may not provide an optimal immune response for dealing withlater stages of infection. Adequate control during the later stages ofinfection may require T cells which are specific for the particularantigens which are expressed at that time.

Post-exposure vaccines which directly target the dormant persistentbacteria may aid in protecting against TB reactivation, therebyenhancing TB control, or even enabling clearance of the infection. Avaccine targeting latent TB could therefore significantly andeconomically reduce global TB infection rates.

Subunit vaccines based on late stage antigens could also be utilised incombination with early stage antigens to provide a multiphase vaccine.Alternatively, late stage antigens could be used to complement andimprove BCG vaccination (either by boosting the BCG response or throughthe development of advanced recombinant BCG strains).

Rv2386c, also known as Mbtl, catalyses the initial transformation inmycobactin biosynthesis by converting chorismate to salicylate (Zwahlenet al. Biochemistry 2007 46:954-964). Rv2386 has previously beenidentified as being associated with expression under stress conditionsalthough did not result in protection when administered as a DNA vaccinein a guinea pig model (Vipond et al. Vaccine 2006 24:6340-6350).

Recently, a range of M. tuberculosis vaccine candidates have beenproposed based on a bioinformatics analysis of the whole genome M.tuberculosis genome (Zvi et al. BMC Medical Genetics 2008 1:18) and onthe testing of differentially expressed proteins in actively andlatently infected individuals (Schuck S D et al. PLoS ONE 20094(5):e5590).

While macrophages have been shown to act as the principal effectors ofMycobacterium immunity, T cells are the predominant inducers of suchimmunity. The essential role of T cells in protection againsttuberculosis is illustrated by the increased rates of TB reactivation inhuman immunodeficiency virus infected individuals, due to the associateddepletion of CD4+ T cells. Furthermore, adoptive transfer of CD4+ Tcells taken at the height of the primary immune response to M.tuberculosis has been shown to confer protection against M. tuberculosisin T cell deficient mice (Orme et al J. Exp. Med. 1983 158:74-83).

Mycobacterium-reactive CD4+ T cells have been shown to be potentproducers of γ-interferon (IFN-γ), which, in turn, has been shown totrigger the anti-mycobacterial effects of macrophages in mice (Flynn etal. J. Exp. Med. 1993 178:2249-2254). While the role of IFN-γ in humansis less clear, studies have shown that 1,25-dihydroxy-vitamin D3, eitheralone or in combination with IFN-γ or tumor necrosis factor-alpha,activates human macrophages to inhibit M. tuberculosis infection.Furthermore, it is known that IFN-γ stimulates human macrophages to make1,25-dihydroxy-vitamin D3. Similarly, interleukin-12 (IL-12) has beenshown to play a role in stimulating resistance to M. tuberculosisinfection. For a review of the immunology of M. tuberculosis infection,see Chan & Kaufmann, Tuberculosis: Pathogenesis, Protection and Control(Bloom ed., 1994), Tuberculosis (2nd ed., Rom and Garay, eds., 2003),and Harrison's Principles of Internal Medicine, Chapter 150, pp. 953-966(16th ed., Braunwald, et al., eds., 2005).

The present invention relates generally to the identification of Rv2386cas a TB antigen (in particular an antigen associated with latent TB) andto related methods and uses in the prevention and treatment of TB,especially the prevention and treatment of latent TB and the preventionor delay of TB reactivation.

The invention therefore provides an Rv2386c protein, variant thereof orimmunogenic fragment thereof, or a polynucleotide encoding said protein,variant or fragment, for use in the treatment or prevention of TB.Suitably, the use may be specifically in the prevention and treatment oflatent TB (especially the treatment of latent TB). Alternatively, theuse may be in the prevention or delay of TB reactivation (especially thedelay of TB reactivation, for example by a period of months, years oreven indefinitely).

The term “Mycobacterium species of the tuberculosis complex” includesthose species traditionally considered as causing the diseasetuberculosis, as well as Mycobacterium environmental and opportunisticspecies that cause tuberculosis and lung disease in immune compromisedpatients, such as patients with AIDS, e.g., M. tuberculosis, M. bovis,or M. africanum, BCG, M. avium, M. intracellulare, M. celatum, M.genavense, M. haemophilum, M. kansasii, M. simiae, M. vaccae, M.fortuitum, and M. scrofulaceum (see, e.g., Harrison's Principles ofInternal Medicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, etal., eds., 2005). The present invention is particularly directed toinfection with M. tuberculosis.

The term “active infection” refers to an infection (e.g. infection by M.tuberculosis) with manifested disease symptoms and/or lesions (suitablywith manifested disease symptoms).

The terms “inactive infection”, “dormant infection” or “latentinfection” refer to an infection (e.g. infection by M. tuberculosis)without manifested disease symptoms and/or lesions (suitably withoutmanifested disease symptoms).

The term “primary tuberculosis” refers to clinical illness(manifestation of disease symptoms) directly following infection (e.g.infection by M. tuberculosis). See, Harrison's Principles of InternalMedicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds.,2005).

The terms “secondary tuberculosis” or “postprimary tuberculosis” referto the reactivation of a dormant, inactive or latent infection (e.g.infection by M. tuberculosis). See, Harrison's Principles of InternalMedicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds.,2005).

The term “tuberculosis reactivation” refers to the later manifestationof disease symptoms in an individual that tests positive for infection(e.g. in a tuberculin skin test, suitably in an in vitro T cell basedassay) test but does not have apparent disease symptoms. The positivediagnostic test indicates that the individual is infected, however, theindividual may or may not have previously manifested active diseasesymptoms that had been treated sufficiently to bring the tuberculosisinto an inactive or latent state. It will be recognised that methods forthe prevention, delay or treatment of tuberculosis reactivation can beinitiated in an individual manifesting active symptoms of disease.

The term “drug resistant” tuberculosis refers to an infection (e.g.infection by M. tuberculosis) wherein the infecting strain is not heldstatic or killed (i.e. is resistant to) one or more of so-called“front-line” chemotherapeutic agents effective in treating tuberculosis(e.g., isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide).

The term “multi-drug resistant” tuberculosis refers to an infection(e.g. infection by M. tuberculosis) wherein the infecting strain isresistant to two or more of “front-line” chemotherapeutic agentseffective in treating tuberculosis.

A “chemotherapeutic agent” refers to a pharmacological agent known andused in the art to treat tuberculosis (e.g. infection by M.tuberculosis). Exemplified pharmacological agents used to treattuberculosis include, but are not limited to amikacin, aminosalicylicacid, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid,kanamycin, pyrazinamide, rifamycins (i.e., rifampin, rifapentine andrifabutin), streptomycin, ofloxacin, ciprofloxacin, clarithromycin,azithromycin and fluoroquinolones. “First-line” or “Front-line”chemotherapeutic agents used to treat tuberculosis that is not drugresistant include isoniazid, rifampin, ethambutol, streptomycin andpyrazinamide. “Second-line” chemotherapeutic agents used to treattuberculosis that has demonstrated drug resistance to one or more“first-line” drugs include ofloxacin, ciprofloxacin, ethionamide,aminosalicylic acid, cycloserine, amikacin, kanamycin and capreomycin.Such pharmacological agents are reviewed in Chapter 48 of Goodman andGilman's The Pharmacological Basis of Therapeutics, Hardman and Limbirdeds., 2001.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer. Suitably apolypeptide according to the present invention will consist only ofnaturally occurring amino acid residues, especially those amino acidsencoded by the genetic code.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogues refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an α carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. Suitably an aminoacid is a naturally occurring amino acid or an amino acid analogue,especially a naturally occurring amino acid and in particular thoseamino acids encoded by the genetic code.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs). Suitably the term “nucleic acid” refers tonaturally occurring deoxyribonucleotides or ribonucleotides and polymersthereof.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated (suitably it refers to the sequenceexplicitly indicated). Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al.,Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

By the term ‘Rv2386c protein sequence’ as used herein is meant thepolypeptide sequence provided in SEQ ID No: 1 or a homologue thereoffrom a Mycobacterium species of the tuberculosis complex, e.g., aspecies such as M. tuberculosis, M. bovis, or M. africanum, or aMycobacterium species that is environmental or opportunistic and thatcauses opportunistic infections such as lung infections in immunecompromised hosts (e.g., patients with AIDS), e.g., BCG, M. avium, M.intracellulare, M. celatum, M. genavense, M. haemophilum, M. kansasii,M. simiae, M. vaccae, M. fortuitum, and M. scrofulaceum (see, e.g.,Harrison's Principles of Internal Medicine, Chapter 150, pp. 953-966,16th ed., Braunwald, et al., eds., 2005).

To ensure a high efficacy rate among vaccinated hosts, the components ofa vaccine should be well conserved among the stains of clinicalsignificance. Suitably, the Rv2386c protein is derived from M.tuberculosis H37Rv (i.e. the polypeptide sequence provided in SEQ IDNo: 1) or a homologue thereof from another M. tuberculosis strain (suchas CDC1551, F11, Haarlem A and C strains). Strains of M. tuberculosiswhich are associated with drug resistance (e.g. MDR or especially XDR)are a particularly valuable basis for the Rv2386c protein sequence.Strains of interest include:

-   -   CDC1551—transmissible and virulent strain    -   Haarlem family (such as Haarlem A)—Drug resistant strains found        in crowded human populations. Members of the Haarlem family        of M. tuberculosis strains have been found in many parts of the        world. The first representative of the family was discovered in        Haarlem, The Netherlands.    -   KZN4207—Drug sensitive isolate from patients in KwaZulu-Natal,        South Africa    -   KZN1435—Multiple drug resistant (MDR) isolate from patients in        KwaZulu-Natal, South Africa    -   KZN605—Extensively drug resistant (XDR) isolate from patients in        KwaZulu-Natal, South Africa    -   C—Highly transmitted in New York City. In one study this strain        was found to be more common among injection drug users and        resistant to reactive nitrogen intermediates (Friedman et al. J.        Infect. Dis. 1997 176(2):478-84)    -   94_M4241A—Isolated in San Francisco in 1994 from a patient born        in China. This strain was previously analysed by genomic        deletion analysis (Gagneux et al., PNAS 2006 103(8):2869-2873).    -   02_1987—Isolated in San Francisco in 2002 from a patient born in        South Korea. This strain was previously analyzed by genomic        deletion analysis (Gagneux et al., PNAS 2006 103(8):2869-2873).    -   T92—Isolated in San Francisco in 1999 from a patient born in The        Philippines. This strain was published in Hirsh et al. PNAS 2004        101:4871-4876).    -   T85—Isolated in San Francisco in 1998 from a patient born in        China. This strain was published in Hirsh et al. PNAS 2004        101:4871-4876).    -   EAS054—Isolated in San Francisco in 1993 from a patient born in        India. This strain was previously analyzed by genomic deletion        analysis (Gagneux et al., PNAS 2006 103(8):2869-2873).

Gagneux et al., PNAS 2006 103(8):2869-2873 and Herbert et al. Infect.Immun. 2007 75(12):5798-5805 provide valuable background on the range ofM. tuberculosis strains which are known to exist.

Most suitably, the Rv2386c protein is selected from the polypeptidesequences provided in SEQ ID Nos: 1 and 3-7, in particular SEQ ID Nos: 1and 3-6, such as SEQ ID No: 1.

Polynucleotides of particular interest are those comprising (such asconsisting of) a sequence encoding:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence.

Polynucleotides will suitably comprise (such as consisting of) a variantof SEQ ID NO: 2 or fragment of SEQ ID NO: 2 which encodes an immunogenicfragment of an Rv2386c protein.

Combinations

The Rv2386c related polypeptides of the present invention can furthercomprise other components designed to enhance their immunogenicity or toimprove these antigens in other respects. For example, improvedisolation of the polypeptide antigens may be facilitated through theaddition of a stretch of histidine residues (commonly known as ahis-tag) towards one end of the antigen.

The term “his-tag” refers to a string of histidine residues, typicallysix residues, that are inserted within the reference sequence. Tominimise disruption of the activity associated with the referencesequence, a his-tag is typically inserted at the N-terminus, usuallyimmediately after the initiating methionine residue, or else at theC-terminus. They are usually heterologous to the native sequence but areincorporated since they facilitate isolation by improving the proteinbinding to immobilised metal affinity chromatography resins (IMAC).Generally speaking the presence or absence of a his-tag is not ofsignificance from the point of view of eliciting a desirable immuneresponse against the reference protein. However, to avoid the risk of anadverse reaction against the his-tag itself, it is considered best tominimise the length of the his-tag e.g. to four or fewer residues, inparticular two residues (or to exclude the use of a his-tag entirely).

To improve the magnitude and/or breadth of the elicited immune responsethe compositions, polypeptides and nucleic acids of the invention cancomprise multiple copies of the inventive antigen and/or additionalheterologous polypeptides (or polynucleotides encoding them) fromMycobacterium species (in particular M. tuberculosis).

One skilled in the art will recognise that when a number of componentsare utilised in combination, the precise presentation can be varied. Forexample, a Rv2386c component and an additional copy of the inventiveantigen or an additional heterologous antigen component could bepresented:

-   -   (1) as two individual polypeptide components;    -   (2) as a fusion protein comprising both polypeptide components;    -   (3) as one polypeptide and one polynucleotide component;    -   (4) as two individual polynucleotide components;    -   (5) as a single polynucleotide encoding two individual        polypeptide components; or    -   (6) as a single polynucleotide encoding a fusion protein        comprising both polypeptide components.

This flexibility applies equally to situations where three or morecomponents are used in combination. However, for convenience, it isoften desirable that when a number of components are present they arecontained within a single fusion protein or a polynucleotide encoding asingle fusion protein. In one embodiment of the invention all antigencomponents are provided as polypeptides (e.g. within a single fusionprotein). In an alternative embodiment of the invention all antigencomponents are provided as polynucleotides (e.g. a singlepolynucleotide, such as one encoding a single fusion protein).

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

“Fusion polypeptide” or “fusion protein” refers to a protein having atleast two heterologous polypeptides (e.g. at least two Mycobacterium sp.polypeptides) covalently linked, either directly or via an amino acidlinker. The polypeptides forming the fusion protein are typically linkedC-terminus to N-terminus, although they can also be linked C-terminus toC-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. Thepolypeptides of the fusion protein can be in any order. This term alsorefers to conservatively modified variants, polymorphic variants,alleles, mutants, immunogenic fragments, and interspecies homologs ofthe antigens that make up the fusion protein. Mycobacterium tuberculosisantigens are described in Cole et al., Nature 393:537 (1998), whichdiscloses the entire Mycobacterium tuberculosis genome. Antigens fromother Mycobacterium species that correspond to M. tuberculosis antigenscan be identified, e.g., using sequence comparison algorithms, asdescribed herein, or other methods known to those of skill in the art,e.g., hybridisation assays and antibody binding assays.

The term “fused” refers to the covalent linkage between two polypeptidesin a fusion protein. The polypeptides are typically joined via a peptidebond, either directly to each other or via an amino acid linker.Optionally, the peptides can be joined via non-peptide covalent linkagesknown to those of skill in the art.

Exemplary M. tuberculosis antigens which may be combined with Rv2386cinclude one or more of (e.g. 1 to 5, such as 1 to 3, in particular 1)the following (such as one or more of (i) to (xii)):

-   -   (i) Mtb8.4 (also known as DPV and Rv1174c), the polypeptide        sequence of which is described in SEQ ID No: 102 of WO97/09428        (cDNA in SEQ ID No: 101) and in Coler et al Journal of        Immunology 1998 161:2356-2364. Of particular interest is the        mature Mtb8.4 sequence which is absent the leading signal        peptide (i.e. amino acid residues 15-96 from SEQ ID No: 102 of        WO97/09428). The full-length polypeptide sequence of Mtb8.4 is        shown in SEQ ID No: 8;    -   (ii) Mtb9.8 (also known as MSL and Rv0287), the polypeptide        sequence of which is described in SEQ ID No: 109 of WO98/53075        (fragments of MSL are disclosed in SEQ ID Nos: 110-124 of        WO98/53075, SEQ ID Nos: 119 and 120 being of particular        interest) and also in Coler et al Vaccine 2009 27:223-233 (in        particular the reactive fragments shown in FIG. 2 therein). The        full-length polypeptide sequence for Mtb9.8 is shown in SEQ ID        No: 9;    -   (iii) Mtb9.9 (also known as Mtb9.9A, MTI, MTI-A and Rv1793) the        polypeptide sequence of which is described in SEQ ID No: 19 of        WO98/53075 and in Alderson et al Journal of Experimental        Medicine 2000 7:551-559 (fragments of MTI are disclosed in SEQ        ID Nos: 17 and 51-66 of WO98/53075, SEQ ID Nos: 17, 51, 52, 53,        56 and 62-65 being of particular interest). A number of        polypeptide variants of MTI are described in SEQ ID Nos: 21, 23,        25, 27, 29 and 31 of WO98/53075 and in Alderson et al Journal of        Experimental Medicine 2000 7:551-559. The full-length        polypeptide sequence for Mtb9.9 is shown in SEQ ID No: 10;    -   (iv) Ra12 (also known as Mtb32A C-terminal antigen) the        polypeptide sequence of which is described in SEQ ID No: 10 of        WO01/98460 and in Skeiky et al Journal of Immunology 2004        172:7618-7682. The full-length polypeptide sequence for Ra12 is        shown in SEQ ID No: 11;    -   (v) Ra35 (also known as Mtb32A N-terminal antigen) the        polypeptide sequence of which is described in SEQ ID No: 8 of        WO01/98460 and in Skeiky et al Journal of Immunology 2004        172:7618-7682. The full-length polypeptide sequence for Ra35 is        shown in SEQ ID No: 12;    -   (vi) TbH9 (also known as Mtb39, Mtb39A, TbH9FL and Rv1196) the        polypeptide sequence of which is described in SEQ ID No: 107 of        WO97/09428, and also in Dillon et al Infection and Immunity 1999        67(6):2941-2950 and Skeiky et al Journal of Immunology 2004        172:7618-7682. The full-length polypeptide sequence for TbH9 is        shown in SEQ ID No: 13;    -   (vii) Mtb40 (also known as HTCC1 and Rv3616c) the polypeptide        sequence of which is described in SEQ ID No: 138 of WO98/53075        (cDNA in SEQ ID No: 137). The full-length polypeptide sequence        for Mtb40 is shown in SEQ ID No: 14;    -   (viii) Mtb41 (also known as MTCC2 and Rv0915c) the polypeptide        sequence of which is described in SEQ ID No: 142 of WO98/53075        (cDNA in SEQ ID No: 140) and in Skeiky et al Journal of        Immunology 2000 165:7140-7149. The full-length polypeptide        sequence for Mtb41 is shown in SEQ ID No: 15;    -   (ix) ESAT-6 (also known as esxA and Rv3875) the polypeptide        sequence of which is described in SEQ ID No: 103 of WO97/09428        (cDNA in SEQ ID No: 104) and in Sorensen et al Infection and        Immunity 1995 63(5):1710-1717. The full-length polypeptide        sequence for ESAT-6 is shown in SEQ ID No: 16;

(x) Ag85 complex antigens (e.g. Ag85A, also known as fbpA and Rv3804c;or Ag85B, also known as fbpB and Rv1886c) which are discussed, forexample, in Content et al Infection and Immunity 1991 59:3205-3212 andin Huygen et al Nature Medicine 1996 2(8):893-898. The full-lengthpolypeptide sequence for Ag85A is shown in SEQ ID No: 17 (the matureprotein of residues 43-338, i.e. lacking the signal peptide, being ofparticular interest). The full-length polypeptide sequence for Ag85B isshown in SEQ ID No: 18 (the mature protein of residues 41-325, i.e.lacking the signal peptide, being of particular interest);

-   -   (xi) Alpha-crystallin (also known as hspX and Rv2031c) which is        described in Verbon et al Journal of Bacteriology 1992        174:1352-1359 and Friscia et al Clinical and Experimental        Immunology 1995 102:53-57 (of particular interest are the        fragments corresponding to residues 71-91, 21-40, 91-110 and        111-130). The full-length polypeptide sequence for        alpha-crystallin is shown in SEQ ID No: 19;    -   (xii) Mpt64 (also known as Rv1980c) which is described in Roche        et al Scandinavian Journal of Immunology 1996 43:662-670. The        full-length polypeptide sequence for MPT64 is shown in SEQ ID        No: 20 (the mature protein of residues 24-228, i.e. lacking the        signal peptide, being of particular interest):    -   (xiii) Mtb32A, the polypeptide sequence of which is described in        SEQ ID No: 2 (full-length) and residues 8-330 of SEQ ID No: 4        (mature) of WO01/98460, especially variants having at least one        of the catalytic triad mutated (e.g. the catalytic serine        residue, which may for example be mutated to alanine). The        full-length polypeptide sequence for Mtb32A is shown in SEQ ID        No: 21. The mature form of Mtb32A having a Ser/Ala mutation is        shown in SEQ ID No: 22;    -   (xiv) TB10.4, the full-length polypeptide sequence for TB10.4 is        shown in SEQ ID No: 23;    -   (xv) Rv1753c, the full-length polypeptide sequence for Rv1753c        from Mycobacterium tuberculosis H37Rv is shown in SEQ ID No:        155; and/or    -   (xvi) Rv2707c, the full-length polypeptide sequence for Rv2707c        from Mycobacterium tuberculosis H37Rv is shown in SEQ ID No:        156.        or combinations thereof, such as (for example combinations such        as (a) to (g)):    -   (a) a combination of Ra12, TbH9 and Ra35 components, for example        in the form of a fusion protein, such as Mtb72f. The polypeptide        sequence of Mtb72f is described in SEQ ID No: 6 of WO2006/117240        (cDNA in SEQ ID No: 5) and in Skeiky et al Journal of Immunology        2004 172:7618-7682 (where it incorporates an optional His-tag to        aid purification, when utilised in the present invention        suitably Mtb72f is absent the optional histidine residues). The        polypeptide sequence for Mtb72f is shown in SEQ ID No: 24;    -   (b) a combination of Ra12, TbH9 and Ser/Ala mutated Ra35 (i.e.        where the catalytic serine residue has been replaced with        alanine) components, for example in the form of a fusion        protein, such as M72. The polypeptide sequence of M72 is        described in SEQ ID No: 4 of WO2006/117240 (cDNA in SEQ ID        No: 3) where it incorporates an optional double histidine to aid        manufacture, when utilised in the present invention M72 may also        incorporate a double histidine though suitably M72 is absent the        optional double histidine (i.e. residues 4-725 from SEQ ID No: 4        of WO2006/117240 are of particular interest). The polypeptide        sequence for M72 is shown in SEQ ID No: 25;    -   (c) a combination of Mtb8.4, Mtb9.8, Mtb9.9 and Mtb41        components, for example in the form of a fusion protein, such as        Mtb71f. The polypeptide sequence of Mtb71f is described in SEQ        ID No: 16 of WO99/051748 (cDNA in SEQ ID No: 15), where it        incorporates an optional His-tag to aid purification, when        utilised in the present invention suitably Mtb71f corresponds to        amino acid residues 9-710 of SEQ ID No: 16 from WO99/051748. The        polypeptide sequence for Mtb71f is shown in SEQ ID No: 26;    -   (d) a combination of Mtb72f or M72 (suitably without optional        histidine residues to aid expression) with Mtb9.8 and Mtb9.9,        for example in a fusion protein. The polypeptide sequence for an        M72-Mtb9.9-Mtb9.8 fusion is shown in SEQ ID No: 27 (M92 fusion),        when used in the present invention, the M72-Mtb9.9-Mtb9.8 fusion        may optionally incorporate a double histidine following the        initiating methionine residue to aid manufacture;    -   (e) a combination of Mtb72f or M72 (suitably without optional        histidine residues to aid expression) with Ag85B, for example in        a fusion protein, such Mtb103f. The polypeptide sequence of        Mtb103f is described in SEQ ID No: 18 of WO03/070187 (cDNA in        SEQ ID No: 10), where it incorporates an optional His-tag to aid        purification, when utilised in the present invention suitably        Mtb103f corresponds to amino acid residues 8-1016 of SEQ ID No:        18 from WO03/070187. Also of particular interest is M103, i.e.        Mtb103f incorporating a Ser/Ala mutation in the Ra35 component,        when utilised in the present invention suitably M103 corresponds        to amino acid residues 8-1016 of SEQ ID No: 18 from WO03/070187        wherein the Ser residue at position 710 has been replaced with        Ala. The polypeptide sequence for M103 is shown in SEQ ID No:        28, when used in the present invention, the M72-Mtb9.9-Mtb9.8        fusion may optionally incorporate a double histidine following        the initiating methionine residue to aid manufacture;    -   (f) a combination of Mtb72f or M72 (suitably without optional        histidine residues to aid expression) with Mtb41, for example in        a fusion protein, such Mtb114f. The polypeptide sequence of        Mtb114f is described in SEQ ID No: 16 of WO03/070187 (cDNA in        SEQ ID No: 9), where it incorporates an optional His-tag to aid        purification, when utilised in the present invention suitably        Mtb114f corresponds to amino acid residues 8-1154 of SEQ ID No:        16 from WO03/070187. Also of particular interest is M114, i.e.        Mtb114f incorporating a Ser/Ala mutation in the Ra35 component,        when utilised in the present invention suitably M114 corresponds        to amino acid residues 8-1154 of SEQ ID No: 16 from WO03/070187        wherein the Ser residue at position 710 has been replaced with        Ala. The polypeptide sequence for M114 is shown in SEQ ID No:        29, when used in the present invention, the M72-Mtb9.9-Mtb9.8        fusion may optionally incorporate a double histidine following        the initiating methionine residue to aid manufacture;    -   (g) a combination of Ag85B and ESAT-6 components, such as in a        fusion described in Doherty et al Journal of Infectious Diseases        2004 190:2146-2153; and/or    -   (h) a combination of Ag85B and TB10.4 components, such as in a        fusion described in Dietrich et al Journal of Immunology 2005        174(10):6332-6339 190:2146-2153.

Combinations of an Rv2386c component and an Mtb40 component are ofparticular interest. Obviously such combinations could optionallycontain other additional antigen components (e.g. an M72 component).

Another combination of interest comprises an Rv2386c component and anM72 component.

A further combination of interest comprises an Rv2386c component and anRv1753c component.

Other combinations of interest include those comprising an Rv2386ccomponent and an Rv2707c component.

An additional combination of interest comprises an Rv2386c component andan alpha-crystallin component.

The skilled person will recognise that combinations need not rely uponthe specific sequences described in above in (i)-(xvi) and (a)-(h), andthat conservatively modified variants (e.g. having at least 70%identity, such as at least 80% identity, in particular at least 90%identity and especially at least 95% identity) or immunogenic fragments(e.g. at least 20% of the full length antigen, such as at least 50% ofthe antigen, in particular at least 70% and especially at least 80%) ofthe described sequences can be used to achieve the same practicaleffect.

Each of the above individual antigen sequences is also disclosed in Coleet al Nature 1998 393:537-544 and Camus Microbiology 2002 148:2967-2973.The genome of M. tuberculosis H37Rv is publicly available, for exampleat the Welcome Trust Sanger Institute website (available on theworldwide web at sanger.ac.uk/Projects/M_tuberculosis/) and elsewhere.

Many of the above antigens are also disclosed in U.S. patent applicationSer. Nos. 08/523,435, 08/523,436, 08/658,800, 08/659,683, 08/818,111,08/818,112, 08/942,341, 08/942,578, 08/858,998, 08/859,381, 09/056,556,09/072,596, 09/072,967, 09/073,009, 09/073,010, 09/223,040, 09/287,849and in PCT patent applications PCT/US98/10407, PCT/US98/10514,PCT/US99/03265, PCT/US99/03268, PCT/US99/07717, WO97/09428 andWO97/09429, WO98/16645, WO98/16646, each of which is herein incorporatedby reference.

The compositions, polypeptides, and nucleic acids of the invention canalso comprise additional polypeptides from other sources. For example,the compositions and fusion proteins of the invention can includepolypeptides or nucleic acids encoding polypeptides, wherein thepolypeptide enhances expression of the antigen, e.g., NS1, an influenzavirus protein (see, e.g. WO99/40188 and WO93/04175). The nucleic acidsof the invention can be engineered based on codon preference in aspecies of choice, e.g., humans (in the case of in vivo expression) or aparticular bacterium (in the case of polypeptide production).

The Rv2386c component may also be administered with one or morechemotherapeutic agents effective against tuberculosis (e.g. M.tuberculosis infection). Examples of such chemotherapeutic agentsinclude, but are not limited to, amikacin, aminosalicylic acid,capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, kanamycin,pyrazinamide, rifamycins (i.e., rifampin, rifapentine and rifabutin),streptomycin, ofloxacin, ciprofloxacin, clarithromycin, azithromycin andfluoroquinolones. Such chemotherapy is determined by the judgment of thetreating physician using preferred drug combinations. “First-line”chemotherapeutic agents used to treat tuberculosis (e.g. M. tuberculosisinfection) that is not drug resistant include isoniazid, rifampin,ethambutol, streptomycin and pyrazinamide. “Second-line”chemotherapeutic agents used to treat tuberculosis (e.g. M. tuberculosisinfection) that has demonstrated drug resistance to one or more“first-line” drugs include ofloxacin, ciprofloxacin, ethionamide,aminosalicylic acid, cycloserine, amikacin, kanamycin and capreomycin.

Conventional chemotherapeutic agents are generally administered over arelatively long period (ca. 9 months). Combination of conventionalchemotherapeutic agents with the administration of a Rv2386c componentaccording to the present invention may enable the chemotherapeutictreatment period to be reduced (e.g. to 8 months, 7 months, 6 months, 5months, 4 months, 3 months or less) without a decrease in efficacy.

Of particular interest is the use of an Rv2386c component in conjunctionwith Bacillus Calmette-Guerin (BCG). For example, in the form of amodified BCG which recombinantly expresses Rv2386c (or a variant orfragment thereof as described herein). Alternatively, the Rv2386ccomponent may be used to enhance the response of a subject to BCGvaccination, either by co-administration or by boosting a previous BCGvaccination. When used to enhance the response of a subject to BCGvaccination, the Rv2386c component may obviously be provided in the formof a polypeptide or a polynucleotide (optionally in conjunction withadditional antigenic components as described above).

The skilled person will recognise that combinations of components neednot be administered together and may be applied: separately or incombination; at the same time, sequentially or within a short period;though the same or through different routes. Nevertheless, forconvenience it is generally desirable (where administration regimes arecompatible) to administer a combination of components as a singlecomposition.

The polypeptides, polynucleotides and compositions of the presentinvention will usually be administered to humans, but are effective inother mammals including domestic mammals (e.g., dogs, cats, rabbits,rats, mice, guinea pigs, hamsters, chinchillas) and agricultural mammals(e.g., cows, pigs, sheep, goats, horses).

Immunogenic Fragments

T cell epitopes are short contiguous stretches of amino acids which arerecognised by T cells (e.g. CD4+ or CD8+ T cells). Identification of Tcell epitopes may be achieved through epitope mapping experiments whichare well known to the person skilled in the art (see, for example, Paul,Fundamental Immunology, 3rd ed., 243-247 (1993); Beiβbarth et alBioinformatics 2005 21(Suppl. 1):i29-i37).

Alternatively, epitopes may be predicted using the approaches discussedin the Examples.

As a result of the crucial involvement of the T cell response intuberculosis, it is readily apparent that fragments of the full lengthRv2386c polypeptide which contain at least one T cell epitope will beimmunogenic and may contribute to immunoprotection. Such fragments arereferred to herein as immunogenic fragments.

Immunogenic fragments according to the present invention will typicallycomprise at least 9 contiguous amino acids from the full lengthpolypeptide sequence (e.g. at least 10), such as at least 12 contiguousamino acids (e.g. at least 15 or at least 20 contiguous amino acids), inparticular at least 50 contiguous amino acids, such as at least 100contiguous amino acids (for example at least 200 contiguous aminoacids). Suitably the immunogenic fragments will be at least 20%, such asat least 50%, at least 70% or at least 80% of the length of the fulllength polypeptide sequence.

It will be understood that in a diverse out-bred population, such ashumans, different HLA types mean that specific epitopes may not berecognised by all members of the population. Consequently, to maximisethe level of recognition and scale of immune response to a polypeptide,it is generally desirable that an immunogenic fragment contains aplurality of the epitopes from the full length sequence (suitably allepitopes).

Particular fragments of the Rv2386c protein which may be of use includethose containing at least one CD4+ epitope, suitably at least two CD4+epitopes and especially all CD4+ epitopes (such as those epitopesdescribed in the Examples and in SEQ ID Nos: 30-52, particularly thoseassociated with a plurality of HLA alleles, e.g. those associated with2, 3, 4, 5 or more alleles).

Other fragments of the Rv2386c protein which may be of use include thosecontaining at least one CD8 epitope, suitably at least two CD8 epitopesand especially all CD8 epitopes (such as those epitopes described in theExamples and in SEQ ID Nos: 53-154, particularly those associated with aplurality of HLA alleles, e.g. those associated with 2, 3, 4, 5 or morealleles).

Where an individual fragment of the full length polypeptide is used,such a fragment is considered to be immunogenic where it elicits aresponse which is at least 20%, suitably at least 50% and especially atleast 75% (such as at least 90%) of the activity of the referencesequence in an in vitro restimulation assay of PBMC or whole blood withspecific antigens (e.g. restimulation for a period of between severalhours to up to two weeks, such as up to one day, 1 day to 1 week or 1 to2 weeks) that measures the activation of the cells vialymphoproliferation, production of cytokines in the supernatant ofculture (measured by ELISA, CBA etc) or characterisation of T and B cellresponses by intra and extracellular staining (e.g. using antibodiesspecific to immune markers, such as CD3, CD4, CD8, IL2, TNFα, IFNγ,CD40L, CD69 etc) followed by analysis with a flowcytometer. Suitably, afragment is considered to be immunogenic where it elicits a responsewhich is at least 20%, suitably at least 50% and especially at least 75%(such as at least 90%) of the activity of the reference sequence in a Tcell proliferation and/or IFN-gamma production assay.

In some circumstances a plurality of fragments of the full lengthpolypeptide (which may or may not be overlapping and may or may notcover the entirety of the full length sequence) may be used to obtain anequivalent biological response to the full length sequence itself. Forexample, at least two immunogenic fragments (such as three, four orfive) as described above, which in combination provide at least 50%,suitably at least 75% and especially at least 90% activity of thereference sequence in an in vitro restimulation assay of PBMC or wholeblood (e.g. a T cell proliferation and/or IFN-gamma production assay).

Variants

“Variants” or “conservatively modified variants” applies to both aminoacid and nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences.

Due to the degeneracy of the genetic code, a large number offunctionally identical nucleic acids encode any given protein. Forinstance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations lead to “silent” or “degenerate” variants, which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognise that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

A polynucleotide of the invention may contain a number of silentvariations (for example, 1-50, such as 1-25, in particular 1-5, andespecially 1 codon(s) may be altered) when compared to the referencesequence. A polynucleotide of the invention may contain a number ofnon-silent conservative variations (for example, 1-50, such as 1-25, inparticular 1-5, and especially 1 codon(s) may be altered) when comparedto the reference sequence. Non-silent variations are those which resultin a change in the encoded amino acid sequence (either though thesubstitution, deletion or addition of amino acid residues). Thoseskilled in the art will recognise that a particular polynucleotidesequence may contain both silent and non-silent conservative variations.

In respect of variants of a protein sequence, the skilled person willrecognise that individual substitutions, deletions or additions topolypeptide, which alters, adds or deletes a single amino acid or asmall percentage of amino acids is a “conservatively modified variant”where the alteration(s) results in the substitution of an amino acidwith a functionally similar amino acid or thesubstitution/deletion/addition of residues which do not substantiallyimpact the biological function of the variant.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

A polypeptide of the invention may contain a number of conservativesubstitutions (for example, 1-50, such as 1-25, in particular 1-10, andespecially 1 amino acid residue(s) may be altered) when compared to thereference sequence. In general, such conservative substitutions willfall within one of the amino-acid groupings specified below, though insome circumstances other substitutions may be possible withoutsubstantially affecting the immunogenic properties of the antigen. Thefollowing eight groups each contain amino acids that are typicallyconservative substitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M).

Suitably such substitutions do not occur in the region of an epitope,and do not therefore have a significant impact on the immunogenicproperties of the antigen.

Protein variants may also include those wherein additional amino acidsare inserted compared to the reference sequence, for example, suchinsertions may occur at 1-10 locations (such as 1-5 locations, suitably1 or 2 locations, in particular 1 location) and may, for example,involve the addition of 50 or fewer amino acids at each location (suchas 20 or fewer, in particular 10 or fewer, especially 5 or fewer).Suitably such insertions do not occur in the region of an epitope, anddo not therefore have a significant impact on the immunogenic propertiesof the antigen. One example of insertions includes a short stretch ofhistidine residues (e.g. 2-6 residues) to aid expression and/orpurification of the antigen in question.

Protein variants include those wherein amino acids have been deletedcompared to the reference sequence, for example, such deletions mayoccur at 1-10 locations (such as 1-5 locations, suitably 1 or 2locations, in particular 1 location) and may, for example, involve thedeletion of 50 or fewer amino acids at each location (such as 20 orfewer, in particular 10 or fewer, especially 5 or fewer). Suitably suchdeletions do not occur in the region of an epitope, and do not thereforehave a significant impact on the immunogenic properties of the antigen.

The skilled person will recognise that a particular protein variant maycomprise substitutions, deletions and additions (or any combinationthereof).

Methods of determining the epitope regions of an antigen are describedand exemplified in the Examples.

Variants preferably exhibit at least about 70% identity, more preferablyat least about 80% identity and most preferably at least about 90%identity (such as at least about 95%, at least about 98% or at leastabout 99%) to the associated reference sequence.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or sub-sequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 70% identity, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99%identity over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecompliment of a test sequence. Optionally, the identity exists over aregion that is at least about 25 to about 50 amino acids or nucleotidesin length, or optionally over a region that is 75-100 amino acids ornucleotides in length. Suitably, the comparison is performed over awindow corresponding to the entire length of the reference sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, references to a segment in whicha sequence may be compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned.Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Current Protocols in Molecular Biology(Ausubel et al., eds. 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153 (1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(website at www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as theneighbourhood word score threshold (Altschul et al., supra). Theseinitial neighbourhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The present invention also extends to polynucleotides comprising a firstnucleotide sequence which selectively hybridises under moderatelystringent conditions (such as under highly stringent conditions) to thecomplement of a second nucleotide sequence which encodes a polypeptidecomprising:

-   -   (i) an Rv2386c protein sequence;    -   (ii) a variant of an Rv2386c protein sequence; or    -   (iii) an immunogenic fragment of an Rv2386c protein sequence.

The phrase “highly stringent hybridisation conditions” refers toconditions under which a probe will hybridise to its target subsequence,typically in a complex mixture of nucleic acid, but to no othersequences. Highly stringent conditions are sequence-dependent and willbe different in different circumstances. Longer sequences hybridisespecifically at higher temperatures. An extensive guide to thehybridisation of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridisation and the strategy of nucleicacid assays” (1993). Generally, highly stringent conditions are selectedto be about 5-10° C. lower than the thermal melting point (T_(m)) forthe specific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridise to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Highly stringent conditions will be those in which thesalt concentration is less than about 1.0 M sodium ion, typically about0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g., 10to 50 nucleotides) and at least about 60° C. for long probes (e.g.,greater than 50 nucleotides). Highly stringent conditions may also beachieved with the addition of destabilising agents such as formamide.For selective or specific hybridisation, a positive signal is at leasttwo times background, optionally 10 times background hybridisation.

Exemplary highly stringent hybridisation conditions can be as following:50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1%SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

Nucleic acids that do not hybridise to each other under highly stringentconditions are still functionally equivalent if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridise under moderately stringent hybridisation conditions.

Exemplary “moderately stringent hybridisation conditions” include ahybridisation in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridisation is at least twicebackground. Those of ordinary skill will readily recognise thatalternative hybridisation and wash conditions can be utilised to provideconditions of similar stringency.

The phrase “selectively (or specifically) hybridises to” refers to thebinding, duplexing, or hybridising of a molecule only to a particularnucleotide sequence under stringent hybridisation conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

In any event, variants of a polypeptide sequence will have essentiallythe same activity as the reference sequence (in the case ofpolynucleotides, variant polynucleotide sequences will encode apolypeptide which has essentially the same activity as the referencesequence). By essentially the same activity is meant at least 50%,suitably at least 75% and especially at least 90% activity of thereference sequence in an in vitro restimulation assay of PBMC or wholeblood with specific antigens (e.g. restimulation for a period of betweenseveral hours to up to two weeks, such as up to one day, 1 day to 1 weekor 1 to 2 weeks) that measures the activation of the cells vialymphoproliferation, production of cytokines in the supernatant ofculture (measured by ELISA, CBA etc) or characterisation of T and B cellresponses by intra and extracellular staining (e.g. using antibodiesspecific to immune markers, such as CD3, CD4, CD8, IL2, TNFa, IFNg,CD40L, CD69 etc) followed by analysis with a flowcytometer. Suitably, byessentially the same activity is meant at least 50%, suitably at least75% and especially at least 90% activity of the reference sequence in aT cell proliferation and/or IFN-gamma production assay.

Polynucleotide Compositions

As used herein, the term “polynucleotide” refers to a molecule that hasbeen isolated free of total genomic DNA of a particular species.Therefore, a polynucleotide encoding a polypeptide refers to apolynucleotide segment that contains one or more coding sequences yet issubstantially isolated away from, or purified free from, total genomicDNA of the species from which the polynucleotide is obtained.

As will be understood by those skilled in the art, the polynucleotidesof this invention can include genomic sequences, extra-genomic andplasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides, peptidesand the like. Such segments may be naturally isolated, or modifiedsynthetically by the hand of man.

“Isolated,” as used herein, means that a polynucleotide is substantiallyaway from other coding sequences, and that the polynucleotide does notcontain large portions of unrelated coding DNA, such as largechromosomal fragments or other functional genes or polypeptide codingregions. An isolated nucleic acid is separated from other open readingframes that flank the gene and encode proteins other than the gene. Ofcourse, this refers to the DNA segment as originally isolated, and doesnot exclude genes or coding regions later added to the segment by thehand of man.

As will be recognised by the skilled artisan, polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. RNA molecules includeHnRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a Mycobacterium antigen or a portion thereof) ormay comprise a variant, or a biological or functional equivalent of sucha sequence. Polynucleotide variants may contain one or moresubstitutions, additions, deletions and/or insertions, as furtherdescribed below, preferably such that the immunogenicity of the encodedpolypeptide is not diminished, relative to the reference protein. Theeffect on the immunogenicity of the encoded polypeptide may generally beassessed as described herein.

In additional embodiments, the present invention provides isolatedpolynucleotides and polypeptides comprising various lengths ofcontiguous stretches of sequence identical to or complementary to one ormore of the sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise at least about 30, 40, 50, 75,100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides ofthe reference sequence disclosed herein as well as all intermediatelengths there between. It will be readily understood that “intermediatelengths”, in this context, means any length between the quoted values,such as 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103,etc.; 150, 151, 152, 153, etc.; including all integers through 200-500;500-1,000, and the like.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear relatively low identity to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention, for example polynucleotides that are optimized forhuman and/or primate codon selection. Further, alleles of the genescomprising the polynucleotide sequences provided herein are within thescope of the present invention. Alleles are endogenous genes that arealtered as a result of one or more mutations, such as deletions,additions and/or substitutions of nucleotides. The resulting mRNA andprotein may, but need not, have an altered structure or function.Alleles may be identified using standard techniques (such ashybridisation, amplification and/or database sequence comparison).

Polynucleotide Identification and Characterisation

Polynucleotides may be identified, prepared and/or manipulated using anyof a variety of well established techniques. For example, apolynucleotide may be identified, as described in more detail below, byscreening a microarray of cDNAs. Such screens may be performed, forexample, using a Synteni microarray (Palo Alto, Calif.) according to themanufacturer's instructions (and essentially as described by Schena etal., Proc. Natl. Acad. Sci. USA 93:10614-10619 (1996) and Heller et al.,Proc. Natl. Acad. Sci. USA 94:2150-2155 (1997)). Alternatively,polynucleotides may be amplified from cDNA prepared from cellsexpressing the proteins described herein, such as M. tuberculosis cells.Such polynucleotides may be amplified via polymerase chain reaction(PCR). For this approach, sequence-specific primers may be designedbased on the sequences provided herein, and may be purchased orsynthesised.

An amplified portion of a polynucleotide may be used to isolate a fulllength gene from a suitable library (e.g., a M. tuberculosis cDNAlibrary) using well known techniques. Within such techniques, a library(cDNA or genomic) is screened using one or more polynucleotide probes orprimers suitable for amplification. Preferably, a library issize-selected to include larger molecules. Random primed libraries mayalso be preferred for identifying 5′ and upstream regions of genes.Genomic libraries are preferred for obtaining introns and extending 5′sequences.

For hybridisation techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridising filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual (2000)). Hybridisingcolonies or plaques are selected and expanded, and the DNA is isolatedfor further analysis. cDNA clones may be analyzed to determine theamount of additional sequence by, for example, PCR using a primer fromthe partial sequence and a primer from the vector. Restriction maps andpartial sequences may be generated to identify one or more overlappingclones. The complete sequence may then be determined using standardtechniques, which may involve generating a series of deletion clones.The resulting overlapping sequences can then be assembled into a singlecontiguous sequence. A full length cDNA molecule can be generated byligating suitable fragments, using well known techniques.

Alternatively, there are numerous amplification techniques for obtaininga full length coding sequence from a partial cDNA sequence. Within suchtechniques, amplification is generally performed via PCR. Any of avariety of commercially available kits may be used to perform theamplification step. Primers may be designed using, for example, softwarewell known in the art. Primers are preferably 22-30 nucleotides inlength, have a GC content of at least 50% and anneal to the targetsequence at temperatures of about 68° C. to 72° C. The amplified regionmay be sequenced as described above, and overlapping sequences assembledinto a contiguous sequence.

One such amplification technique is inverse PCR (see Triglia et al.,Nucl. Acids Res. 16:8186 (1988)), which uses restriction enzymes togenerate a fragment in the known region of the gene. The fragment isthen circularised by intramolecular ligation and used as a template forPCR with divergent primers derived from the known region. Within analternative approach, sequences adjacent to a partial sequence may beretrieved by amplification with a primer to a linker sequence and aprimer specific to a known region. The amplified sequences are typicallysubjected to a second round of amplification with the same linker primerand a second primer specific to the known region. A variation on thisprocedure, which employs two primers that initiate extension in oppositedirections from the known sequence, is described in WO 96/38591. Anothersuch technique is known as “rapid amplification of cDNA ends” or RACE.This technique involves the use of an internal primer and an externalprimer, which hybridises to a polyA region or vector sequence, toidentify sequences that are 5′ and 3′ of a known sequence. Additionaltechniques include capture PCR (Lagerstrom et al., PCR Methods Applic.1:111-19 (1991)) and walking PCR (Parker et al., Nucl. Acids. Res.19:3055-60 (1991)). Other methods employing amplification may also beemployed to obtain a full length cDNA sequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

Polynucleotide Expression in Host Cells

Polynucleotide sequences or fragments thereof which encode polypeptides,or fusion proteins or functional equivalents thereof, may be used inrecombinant DNA molecules to direct expression of a polypeptide inappropriate host cells. Due to the inherent degeneracy of the geneticcode, other DNA sequences that encode substantially the same or afunctionally equivalent amino acid sequence may be produced and thesesequences may be used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences can be engineered using methodsgenerally known in the art in order to alter polypeptide encodingsequences for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. For example, DNA shuffling by random fragmentation andPCR reassembly of gene fragments and synthetic oligonucleotides may beused to engineer the nucleotide sequences. In addition, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

Natural, modified, or recombinant nucleic acid sequences may be ligatedto a heterologous sequence to encode a fusion protein. For example, toscreen peptide libraries for inhibitors of polypeptide activity, it maybe useful to encode a chimeric protein that can be recognised by acommercially available antibody. A fusion protein may also be engineeredto contain a cleavage site located between the polypeptide-encodingsequence and the heterologous protein sequence, so that the polypeptidemay be cleaved and purified away from the heterologous moiety.

Sequences encoding a desired polypeptide may be synthesised, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. et al., Nucl. Acids Res. Symp. Ser. pp. 215-223 (1980), Horn et al.,Nucl. Acids Res. Symp. Ser. pp. 225-232 (1980)). Alternatively, theprotein itself may be produced using chemical methods to synthesize theamino acid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge et al., Science 269:202-204 (1995)) and automated synthesis maybe achieved, for example, using the ABI 431A Peptide Synthesizer (PerkinElmer, Palo Alto, Calif.).

A newly synthesised peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, Proteins,Structures and Molecular Principles (1983)) or other comparabletechniques available in the art. The composition of the syntheticpeptides may be confirmed by amino acid analysis or sequencing (e.g.,the Edman degradation procedure). Additionally, the amino acid sequenceof a polypeptide, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a desired polypeptide, the nucleotide sequencesencoding the polypeptide, or functional equivalents, may be insertedinto an appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook et al.,Molecular Cloning, A Laboratory Manual (2000), and Ausubel et al.,Current Protocols in Molecular Biology (updated annually).

A variety of expression vector/host systems may be utilised to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilised, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are needed, for example for the inductionof antibodies, vectors which direct high level expression of fusionproteins that are readily purified may be used. Such vectors include,but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of β-galactosidase so that a hybrid protein isproduced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem.264:5503-5509 (1989)); and the like. pGEX Vectors (Promega, Madison,Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. Other vectors containing constitutive orinducible promoters include GAP, PGK, GAL and ADH. For reviews, seeAusubel et al. (supra) and Grant et al., Methods Enzymol. 153:516-544(1987) and Romas et al. Yeast 8 423-88 (1992).

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671-1680(1984); Broglie et al., Science 224:838-843 (1984); and Winter et al.,Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, e.g., Hobbs in McGraw HillYearbook of Science and Technology pp. 191-196 (1992)).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhardet al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad.Sci. U.S.A. 81:3655-3659 (1984)). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells. Methods and protocols for workingwith adenovirus vectors are reviewed in Wold, Adenovirus Methods andProtocols, 1998. Additional references regarding use of adenovirusvectors can be found in Adenovirus: A Medical Dictionary, Bibliography,and Annotated Research Guide to Internet References, 2004.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf. et al., ResultsProbl. Cell Differ. 20:125-162 (1994)).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11:223-32 (1977)) and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817-23 (1990)) geneswhich can be employed in tk.sup.- or aprt.sup.-cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70(1980)); npt, which confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); andals or pat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilise indole in place of tryptophan, or hisD, which allows cells toutilise histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci. U.S.A. 85:8047-51 (1988)). Recently, the use of visiblemarkers has gained popularity with such markers as anthocyanins,β-glucuronidase and its substrate GUS, and luciferase and its substrateluciferin, being widely used not only to identify transformants, butalso to quantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes et al., Methods Mol.Biol. 55:121-131 (1995)).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridisations and protein bioassayor immunoassay techniques which include membrane, solution, or chipbased technologies for the detection and/or quantification of nucleicacid or protein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilising monoclonal antibodies reactive totwo non-interfering epitopes on a given polypeptide may be preferred forsome applications, but a competitive binding assay may also be employed.These and other assays are described, among other places, in Hampton etal., Serological Methods, a Laboratory Manual (1990) and Maddox et al.,J. Exp. Med. 158:1211-1216 (1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labelled hybridisation or PCR probesfor detecting sequences related to polynucleotides includeoligolabeling, nick translation, end-labelling or PCR amplificationusing a labelled nucleotide. Alternatively, the sequences, or anyportions thereof may be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits. Suitable reporter molecules or labels,which may be used include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides may bedesigned to contain signal sequences which direct secretion of theencoded polypeptide through a prokaryotic or eukaryotic cell membrane.Other recombinant constructions may be used to join sequences encoding apolypeptide of interest to nucleotide sequence encoding a polypeptidedomain which will facilitate purification of soluble proteins. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilised immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp.,Seattle, Wash.). The inclusion of cleavable linker sequences such asthose specific for Factor XA or enterokinase (Invitrogen. San Diego,Calif.) between the purification domain and the encoded polypeptide maybe used to facilitate purification. One such expression vector providesfor expression of a fusion protein containing a polypeptide of interestand a nucleic acid encoding 6 histidine residues preceding a thioredoxinor an enterokinase cleavage site. The histidine residues facilitatepurification on IMIAC (immobilised metal ion affinity chromatography) asdescribed in Porath et al., Prot. Exp. Purif. 3:263-281 (1992) while theenterokinase cleavage site provides a means for purifying the desiredpolypeptide from the fusion protein. A discussion of vectors whichcontain fusion proteins is provided in Kroll et al., DNA Cell Biol.12:441-453 (1993).

In Vivo Polynucleotide Delivery Techniques

In additional embodiments, genetic constructs comprising one or more ofthe polynucleotides of the invention are introduced into cells in vivo.This may be achieved using any of a variety or well known approaches,several of which are outlined below for the purpose of illustration.

1. Adenovirus

One of the preferred methods for in vivo delivery of one or more nucleicacid sequences involves the use of an adenovirus expression vector.“Adenovirus expression vector” is meant to include those constructscontaining adenovirus sequences sufficient to (a) support packaging ofthe construct and (b) to express a polynucleotide that has been clonedtherein in a sense or antisense orientation. Of course, in the contextof an antisense construct, expression does not require that the geneproduct be synthesised.

The expression vector comprises a genetically engineered form of anadenovirus. Knowledge of the genetic organisation of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus &Horwitz, Semin. Virol., 3:237-252, 1992). In contrast to retrovirus, theadenoviral infection of host cells does not result in chromosomalintegration because adenoviral DNA can replicate in an episomal mannerwithout potential genotoxicity. Also, adenoviruses are structurallystable, and no genome rearrangement has been detected after extensiveamplification. Adenovirus can infect virtually all epithelial cellsregardless of their cell cycle stage. So far, adenoviral infectionappears to be linked only to mild disease such as acute respiratorydisease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titre, widetarget-cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,Radiother. Oncol., 19(3):197-218, 1990). The products of the late genes,including the majority of the viral capsid proteins, are expressed onlyafter significant processing of a single primary transcript issued bythe major late promoter (MLP). The MLP, (located at 16.8 m.u.) isparticularly efficient during the late phase of infection, and all themRNA's issued from this promoter possess a 5′-tripartite leader (TPL)sequence which makes them preferred mRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al., J.Gen. Virol., 36:59-74, 1977). Since the E3 region is dispensable fromthe adenovirus genome (Jones & Shenk, Cell, 13:181-188, 1978), thecurrent adenovirus vectors, with the help of 293 cells, carry foreignDNA in either the E1, the D3 or both regions (Graham & Prevec, Methodsin Molecular Biology, 7:109-128, 1991). In nature, adenovirus canpackage approximately 105% of the wild-type genome (Ghosh-Choudhury etal., Biochemical and Biophysical Research Communications,147(3):964-973, 1987), providing capacity for about 2 extra kB of DNA.Combined with the approximately 5.5 kB of DNA that is replaceable in theE1 and E3 regions, the maximum capacity of the current adenovirus vectoris under 7.5 kB, or about 15% of the total length of the vector. Morethan 80% of the adenovirus viral genome remains in the vector backboneand is the source of vector-borne cytotoxicity. Also, the replicationdeficiency of the E1-deleted virus is incomplete. For example, leakageof viral gene expression has been observed with the currently availablevectors at high multiplicities of infection (MOI) (Mulligan, Science,260:926-932, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the currently preferred helper cell line is 293.

Racher et al. (Racher et al., Biotechnol. Prog., 11(5):575-583, 1995)have disclosed improved methods for culturing 293 cells and propagatingadenovirus. In one format, natural cell aggregates are grown byinoculating individual cells into 1 liter siliconised spinner flasks(Techne, Cambridge, UK) containing 100-200 ml of medium. Followingstirring at 40 rpm, the cell viability is estimated with trypan blue. Inanother format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5g/l) is employed as follows. A cell inoculum, resuspended in 5 ml ofmedium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask andleft stationary, with occasional agitation, for 1 to 4 h. The medium isthen replaced with 50 ml of fresh medium and shaking initiated. Forvirus production, cells are allowed to grow to about 80% confluence,after which time the medium is replaced (to 25% of the final volume) andadenovirus added at an MOI of 0.05. Cultures are left stationaryovernight, following which the volume is increased to 100% and shakingcommenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain aconditional replication-defective adenovirus vector for use in thepresent invention, since Adenovirus type 5 is a human adenovirus aboutwhich a great deal of biochemical and genetic information is known, andit has historically been used for most constructions employingadenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al. (Karlsson et al., The EMBO Journal,5(9):2377-2385, 1986) or in the E4 region where a helper cell line orhelper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightitres, e.g., 10⁹-10¹¹ plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., Am. Rev. Respir. Dis. 88:394-403,1963; Top et al., J. Infect. Dis., 124(2):148-160, 1971), demonstratingtheir safety and therapeutic potential as in vivo gene transfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., Gene, 101(2):195-202, 1991; Gomez-Foix et al., Biotechniques,34(3):600-602, 1992) and vaccine development (Grunhaus & Horwitz, 1992;Graham & Prevec, 1991). Recently, animal studies suggested thatrecombinant adenovirus could be used for gene therapy(Stratford-Perricaudet & Perricaudet, Science, 252(5004):431-434, 1991;Stratford-Perricaudet et al., Hum. Gene Ther., 1(3):241-256, 1990; Richet al., Hum. Gene Ther., 4(4):461-476, 1993). Studies in administeringrecombinant adenovirus to different tissues include trachea instillation(Rosenfeld et al., Science, 252(5004):431-434, 1991; Rosenfeld et al.,Cell, 68(1):143-155, 1992), muscle injection (Ragot et al., Nature,361(6413):647-650, 1993), peripheral intravenous injections (Herz &Gerard, Proc. Natl. Acad. Sci. USA, 90(7):2812-2816, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al.,Science, 259(5097):988-990, 1993).

Adenovirus vectors may originate from human adenovirus. Alternativelythey may originate from adenovirus of other species e.g. chimpanzeewhich may have the advantage that the viral vectors are not neutralisedby antibodies against human adenovirus circulating in many humansubjects (see e.g.: Tatsis N et al Gene Therapy 2006 13:421-429).

Adenovirus type 35, which is relatively uncommon and therefore there arelow levels of pre-existing immunity to the vector itself, has been usedas a delivery system in certain tuberculosis vaccines which are beingdeveloped (see for example, Radosevic et al Infection and Immunity 200775(8):4105-4115). Adenovirus type 35 may also be of particular value inthe present invention as a delivery vector.

2. Retroviruses

The retroviruses are a group of single-stranded RNA virusescharacterised by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, J. Med.Virol., 31(1):43-49, 1990). The resulting DNA then stably integratesinto cellular chromosomes as a provirus and directs synthesis of viralproteins. The integration results in the retention of the viral genesequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsidproteins, polymerase enzyme, and envelope components, respectively. Asequence found upstream from the gag gene contains a signal forpackaging of the genome into virions. Two long terminal repeat (LTR)sequences are present at the 5′ and 3′ ends of the viral genome. Thesecontain strong promoter and enhancer sequences and are also required forintegration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding oneor more oligonucleotide or polynucleotide sequences of interest isinserted into the viral genome in the place of certain viral sequencesto produce a virus that is replication-defective. In order to producevirions, a packaging cell line containing the gag, pol, and env genesbut without the LTR and packaging components is constructed (Mann etal., Virology, 196(1):57-69, 1983). When a recombinant plasmidcontaining a cDNA, together with the retroviral LTR and packagingsequences is introduced into this cell line (by calcium phosphateprecipitation for example), the packaging sequence allows the RNAtranscript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas &Rubenstein, Biotechnology, 10:493-513, 1988; Temin, Cell Biophys.,9(1-2):9-16, 1986; Mann et al., 1983). The media containing therecombinant retroviruses is then collected, optionally concentrated, andused for gene transfer. Retroviral vectors are able to infect a broadvariety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., Virology,67(1):242-8, 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., Proc. Natl. Acad. Sci. USA., 86(23):9079-9083, 1989). Usingantibodies against major histocompatibility complex class I and class IIantigens, they demonstrated the infection of a variety of human cellsthat bore those surface antigens with an ecotropic virus in vitro (Rouxet al., 1989).

3. Adeno-Associated Viruses

AAV (Ridgeway, FEMS Microbiol. Immunol., (3):157-62, 1988; Hermonat &Muzycska, Proc. Nat. Acad. Sci. USA, 81:6466-6470, 1984) is a parovirus,discovered as a contamination of adenoviral stocks. It is a ubiquitousvirus (antibodies are present in 85% of the US human population) thathas not been linked to any disease. It is also classified as adependovirus, because its replication is dependent on the presence of ahelper virus, such as adenovirus. Five serotypes have been isolated, ofwhich AAV-2 is the best characterised.

AAV has a single-stranded linear DNA that is encapsidated into capsidproteins VP1, VP2 and VP3 to form an icosahedral virion of 20 to 24 nmin diameter (Muzyczka & McLaughlin, J. Virol. 62(6):1963-1973, 1988).

The AAV DNA is approximately 4.7 kilobases long. It contains two openreading frames and is flanked by two ITRs. There are two major genes inthe AAV genome: rep and cap. The rep gene codes for proteins responsiblefor viral replications, whereas cap codes for capsid protein VP1-3. EachITR forms a T-shaped hairpin structure. These terminal repeats are theonly essential cis components of the AAV for chromosomal integration.Therefore, the AAV can be used as a vector with all viral codingsequences removed and replaced by the cassette of genes for delivery.Three viral promoters have been identified and named p5, p19, and p40,according to their map position. Transcription from p5 and p19 resultsin production of rep proteins, and transcription from p40 produces thecapsid proteins (Hermonat & Muzyczka, 1984).

There are several factors that prompted researchers to study thepossibility of using rAAV as an expression vector. One is that therequirements for delivering a gene to integrate into the host chromosomeare surprisingly few. It is necessary to have the 145-bp ITRs, which areonly 6% of the AAV genome. This leaves room in the vector to assemble a4.5-kb DNA insertion. While this carrying capacity may prevent the AAVfrom delivering large genes, it is amply suited for delivering antisenseconstructs.

AAV is also a good choice of delivery vehicles due to its safety. Thereis a relatively complicated rescue mechanism: not only wild typeadenovirus but also AAV genes are required to mobilise rAAV. Likewise,AAV is not pathogenic and not associated with any disease. The removalof viral coding sequences minimises immune reactions to viral geneexpression, and therefore, rAAV does not evoke an inflammatory response.

4. Other Viral Vectors as Expression Constructs

Other viral vectors may be employed as expression constructs in thepresent invention for the delivery of oligonucleotide or polynucleotidesequences to a host cell. Vectors derived from viruses such as vacciniavirus (Ridgeway, 1988; Coupar et al., Gene, 15:68(1):1-10, 1988),lentiviruses, polio viruses and herpes viruses may be employed. Otherpoxvirus derived vectors, such as fowl-pox derived vectors, may also beexpected to be of use. They offer several attractive features forvarious mammalian cells (Friedmann, Mol. Biol. Med., 6(2):117-125, 1989;Ridgeway, 1988; Coupar et al., 1988; Horwich et al., J. Virol.64(2):642-50, 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. (Chang et al., Immunogenetics,33(3):171-177, 1991) introduced the chloramphenicol acetyltransferase(CAT) gene into duck hepatitis B virus genome in the place of thepolymerase, surface, and pre-surface coding sequences. It wascotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titres of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

Additional ‘viral’ vectors include virus like particles (VLPs) andphages.

5. Non-Viral Vectors

In order to effect expression of the oligonucleotide or polynucleotidesequences of the present invention, the expression construct must bedelivered into a cell. This delivery may be accomplished in vitro, as inlaboratory procedures for transforming cells lines, or in vivo or exvivo, as in the treatment of certain disease states. As described above,one preferred mechanism for delivery is via viral infection where theexpression construct is encapsulated in an infectious viral particle.

Once the expression construct has been delivered into the cell thenucleic acid encoding the desired oligonucleotide or polynucleotidesequences may be positioned and expressed at different sites. In certainembodiments, the nucleic acid encoding the construct may be stablyintegrated into the genome of the cell. This integration may be in thespecific location and orientation via homologous recombination (genereplacement) or it may be integrated in a random, non-specific location(gene augmentation). In yet further embodiments, the nucleic acid may bestably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronisationwith the host cell cycle. How the expression construct is delivered to acell and where in the cell the nucleic acid remains is dependent on thetype of expression construct employed.

In certain embodiments of the invention, the expression constructcomprising one or more oligonucleotide or polynucleotide sequences maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed, for example, by any method which physicallyor chemically permeabilises the cell membrane. This is particularlyapplicable for transfer in vitro but it may be applied to in vivo use aswell. Dubensky et al. (Dubensky et al., J. Virol. 50(3):779-83, 1984)successfully injected polyomavirus DNA in the form of calcium phosphateprecipitates into liver and spleen of adult and newborn micedemonstrating active viral replication and acute infection. Benvenisty &Reshef (Benvenisty & Reshef, Proc. Natl. Acad. Sci. USA,83(24):9551-9555, 1986) also demonstrated that direct intraperitonealinjection of calcium phosphate-precipitated plasmids results inexpression of the transfected genes. It is envisioned that DNA encodinga gene of interest may also be transferred in a similar manner in vivoand express the gene product.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., J. Virol., 61(5):1552-1558,1987). Several devices for accelerating small particles have beendeveloped. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., Proc. Natl. Acad. Sci. USA, 87(24):9568-9572, 1990). Themicroprojectiles used have consisted of biologically inert substancessuch as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,FEBS Lett. 287(1-2):118-20, 1991). This may require surgical exposure ofthe tissue or cells, to eliminate any intervening tissue between the gunand the target organ, i.e., ex vivo treatment. Again, DNA encoding aparticular gene may be delivered via this method and still beincorporated.

Bacteria may also be utilised as a delivery method (e.g. listeria, seeWO2004/11048) and in particular BCG.

Polypeptide Compositions

The present invention, in other aspects, provides polypeptidecompositions.

Generally, a polypeptide of the invention will be an isolatedpolypeptide (i.e. separated from those components with which it mayusually be found in nature).

For example, a naturally-occurring protein is isolated if it isseparated from some or all of the coexisting materials in the naturalsystem. Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

Polypeptides may be prepared using any of a variety of well knowntechniques. Recombinant polypeptides encoded by DNA sequences asdescribed above may be readily prepared from the DNA sequences using anyof a variety of expression vectors known to those of ordinary skill inthe art. Expression may be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga DNA molecule that encodes a recombinant polypeptide. Suitable hostcells include prokaryotes, yeast, and higher eukaryotic cells, such asmammalian cells and plant cells. Preferably, the host cells employed areE. coli, yeast or a mammalian cell line such as COS or CHO. Supernatantsfrom suitable host/vector systems which secrete recombinant protein orpolypeptide into culture media may be first concentrated using acommercially available filter. Following concentration, the concentratemay be applied to a suitable purification matrix such as an affinitymatrix or an ion exchange resin. Finally, one or more reverse phase HPLCsteps can be employed to further purify a recombinant polypeptide.

Polypeptides of the invention, immunogenic fragments thereof, and othervariants having less than about 100 amino acids, and generally less thanabout 50 amino acids, may also be generated by synthetic means, usingtechniques well known to those of ordinary skill in the art. Forexample, such polypeptides may be synthesised using any of thecommercially available solid-phase techniques, such as the Merrifieldsolid-phase synthesis method, where amino acids are sequentially addedto a growing amino acid chain. See Merrifield, J. Am. Chem. Soc.85:2149-2146 (1963). Equipment for automated synthesis of polypeptidesis commercially available from suppliers such as Perkin Elmer/AppliedBioSystems Division (Foster City, Calif.), and may be operated accordingto the manufacturer's instructions.

Within certain specific embodiments, a polypeptide may be a fusionprotein that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence, examples of such proteins include tetanus,tuberculosis and hepatitis proteins (see, e.g., Stoute et al., New Engl.J. Med. 336:86-91 (1997)). A fusion partner may, for example, assist inproviding T helper epitopes (an immunological fusion partner),preferably T helper epitopes recognised by humans, or may assist inexpressing the protein (an expression enhancer) at higher yields thanthe native recombinant protein. Certain preferred fusion partners areboth immunological and expression enhancing fusion partners. Otherfusion partners may be selected so as to increase the solubility of theprotein or to enable the protein to be targeted to desired intracellularcompartments. Still further fusion partners include affinity tags, whichfacilitate purification of the protein.

Fusion proteins may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion protein isexpressed as a recombinant protein, allowing the production of increasedlevels, relative to a non-fused protein, in an expression system.Briefly, DNA sequences encoding the polypeptide components may beassembled separately, and ligated into an appropriate expression vector.The 3′ end of the DNA sequence encoding one polypeptide component isligated, with or without a peptide linker, to the 5′ end of a DNAsequence encoding the second polypeptide component so that the readingframes of the sequences are in phase. This permits translation into asingle fusion protein that retains the biological activity of bothcomponent polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No.4,751,180. The linker sequence may generally be from 1 to about 50 aminoacids in length. Linker sequences are not required when the first andsecond polypeptides have non-essential N-terminal amino acid regionsthat can be used to separate the functional domains and prevent stericinterference.

Within preferred embodiments, an immunological fusion partner is derivedfrom protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292 (1986)). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798 (1992)). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

T Cells

Immunotherapeutic compositions may also, or alternatively, comprise Tcells specific for a Mycobacterium antigen. Such cells may generally beprepared in vitro or ex vivo, using standard procedures. For example, Tcells may be isolated from bone marrow, peripheral blood, or a fractionof bone marrow or peripheral blood of a patient, using a commerciallyavailable cell separation system, such as the Isolex™ System, availablefrom Nexell Therapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No.5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO92/07243). Alternatively, T cells may be derived from related orunrelated humans, non-human mammals, cell lines or cultures.

T cells may be stimulated with a polypeptide of the invention,polynucleotide encoding such a polypeptide, and/or an antigen presentingcell (APC) that expresses such a polypeptide. Such stimulation isperformed under conditions and for a time sufficient to permit thegeneration of T cells that are specific for the polypeptide. Preferably,the polypeptide or polynucleotide is present within a delivery vehicle,such as a microsphere, to facilitate the generation of specific T cells.

T cells are considered to be specific for a polypeptide of the inventionif the T cells specifically proliferate, secrete cytokines or killtarget cells coated with the polypeptide or expressing a gene encodingthe polypeptide. T cell specificity may be evaluated using any of avariety of standard techniques. For example, within a chromium releaseassay or proliferation assay, a stimulation index of more than two foldincrease in lysis and/or proliferation, compared to negative controls,indicates T cell specificity. Such assays may be performed, for example,as described in Chen et al., Cancer Res. 54:1065-1070 (1994)).Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labelling cultures of T cells with tritiatedthymidine and measuring the amount of tritiated thymidine incorporatedinto DNA). Contact with a polypeptide of the invention (100 ng/ml-100μg/ml, preferably 200 ng/ml-25 μg/ml) for 3-7 days should result in atleast a two fold increase in proliferation of the T cells. Contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1 (1998)). T cells that have been activated inresponse to a polypeptide, polynucleotide or polypeptide-expressing APCmay be CD4⁺ and/or CD8⁺. Protein-specific T cells may be expanded usingstandard techniques. Within preferred embodiments, the T cells arederived from a patient, a related donor or an unrelated donor, and areadministered to the patient following stimulation and expansion.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to a polypeptide, polynucleotide or APC can be expanded innumber either in vitro or in vivo. Proliferation of such T cells invitro may be accomplished in a variety of ways. For example,

the T cells can be re-exposed to a polypeptide, or a short peptidecorresponding to an immunogenic portion of such a polypeptide, with orwithout the addition of T cell growth factors, such as interleukin-2,and/or stimulator cells that synthesise a polypeptide. Alternatively,one or more T cells that proliferate in the presence of the protein canbe expanded in number by cloning. Methods for cloning cells are wellknown in the art, and include limiting dilution.

Pharmaceutical Compositions

In additional embodiments, the polynucleotide, polypeptide, T-celland/or antibody compositions disclosed herein will be formulated inpharmaceutically-acceptable or physiologically-acceptable solutions foradministration to a cell or an animal, either alone, or in combinationwith one or more other modalities of therapy.

It will also be understood that, if desired, the nucleic acid segment(e.g., RNA or DNA) that expresses a polypeptide as disclosed herein maybe administered in combination with other agents as well, such as, e.g.,other proteins or polypeptides or various pharmaceutically-activeagents, including chemotherapeutic agents effective against a M.tuberculosis infection. In fact, there is virtually no limit to othercomponents that may also be included, given that the additional agentsdo not cause a significant adverse effect upon contact with the targetcells or host tissues. The compositions may thus be delivered along withvarious other agents as required in the particular instance. Suchcompositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesised as describedherein. Likewise, such compositions may further comprise substituted orderivatised RNA or DNA compositions.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation. Other routes ofadministration include via the mucosal surfaces.

Typically, formulations comprising a therapeutically effective amountdeliver about 0.1 ug to about 1000 ug of polypeptide per administration,more typically about 2.5 ug to about 100 ug of polypeptide peradministration. In respect of polynucleotide compositions, thesetypically deliver about 10 ug to about 20 mg of the inventivepolynucleotide per administration, more typically about 0.1 mg to about10 mg of the inventive polynucleotide per administration.

Naturally, the amount of active compound(s) in each therapeuticallyuseful composition may be prepared is such a way that a suitable dosagewill be obtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

1. Oral Delivery

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al.,Nature, 386(6623):410-414, 1997; Hwang et al., Adv. Drug Deliv. Rev.32:139-152, 1998; U.S. Pat. No. 5,641,515; U.S. Pat. No. 5,580,579 andU.S. Pat. No. 5,792,451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, as gum tragacanth,acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate;a disintegrating agent, such as corn starch, potato starch, alginic acidand the like; a lubricant, such as magnesium stearate; and a sweeteningagent, such as sucrose, lactose or saccharin may be added or aflavouring agent, such as peppermint, oil of wintergreen, or cherryflavouring. When the dosage unit form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier. Various othermaterials may be present as coatings or to otherwise modify the physicalform of the dosage unit. For instance, tablets, pills, or capsules maybe coated with shellac, sugar, or both. A syrup of elixir may containthe active component, sucrose as a sweetening agent methyl andpropylparabens as preservatives, a dye and flavouring, such as cherry ororange flavour. Of course, any material used in preparing any dosageunit form should be pharmaceutically pure and substantially non-toxic inthe amounts employed. In addition, the active components may beincorporated into sustained-release preparation and formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

2. Injectable Delivery

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, intradermally, or even intraperitoneallyas described in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 andU.S. Pat. No. 5,399,363 (each specifically incorporated herein byreference in its entirety). Solutions of the active compounds as freebase or pharmacologically acceptable salts may be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions may also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, e.g., Remington's PharmaceuticalSciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

3. Nasal and Buccal Delivery

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, buccal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering genes, nucleic acids, andpeptide compositions directly to the lungs eg via nasal and buccalaerosol sprays has been described e.g., in U.S. Pat. No. 5,756,353 andU.S. Pat. No. 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., J. Control. Release,52(1-2):81-87, 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat.No. 5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

4. Liposome-, Nanocapsule-, and Microparticle-Mediated Delivery

In certain embodiments, the inventors contemplate the use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, or ananoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically-acceptable formulations of the nucleic acids orconstructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art (see for example, Couvreuret al., FEBS Lett. 84(2):323-326, 1977; Couvreur, Crit. Rev. Ther. Drug.Carrier Syst., 5(1):1-20, 1988; Lasic, Trends Biotechnol.,16(7):307-321, 1998; which describes the use of liposomes andnanocapsules in the targeted antibiotic therapy for intracellularbacterial infections and diseases). Recently, liposomes were developedwith improved serum stability and circulation half-times (Gabizon &Papahadjopoulos, Proc. Natl. Acad. Sci. USA 85(18):6949-6953, 1988;Allen and Choun, FEBS Lett., 223:42-46, 1987; U.S. Pat. No. 5,741,516,specifically incorporated herein by reference in its entirety). Further,various methods of liposome and liposome like preparations as potentialdrug carriers have been reviewed (Takakura, Nihon Rinsho.,56(3):691-695, 1998; Chandran et al., Indian J. Exp. Biol.,35(8):801-809, 1997; Margalit, Crit. Rev. Ther. Drug Carrier Syst.,12(2-3):233-261, 1995; U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157;U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S. Pat. No.5,795,587, each specifically incorporated herein by reference in itsentirety).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J. Biol. Chem., 265(27):16337-16342, 1990; Muller et al., DNACell Biol., 9(3):221-229, 1990). In addition, liposomes are free of theDNA length constraints that are typical of viral-based delivery systems.Liposomes have been used effectively to introduce genes, drugs (Heath &Martin, Chem. Phys. Lipids, 40(2-4):347-358, 1986; Heath et al.,Biochim. Biophys. Acta., 862(1):72-80, 1986; Balazsovits et al., CancerChemother. Pharmacol., 23(2):81-86, 1989; Fresta & Puglisi, J. DrugTarget., 4(2):95-101, 1996), radiotherapeutic agents (Pikul et al.,Arch. Surg., 122(12):1417-1420, 1987), enzymes (Imaizumi et al., Stroke,21(9):1312-1317, 1990a; Imaizumi et al., Acta. Neurochir. Suppl. (Wien),51:236-238, 1990b), viruses (Faller & Baltimore, J. Virol.,49(1):269-72, 1984), transcription factors and allosteric effectors(Nicolau & Gersonde, Naturwissenschaften., 66(11):563-566, 1979) into avariety of cultured cell lines and animals. In addition, severalsuccessful clinical trails examining the effectiveness ofliposome-mediated drug delivery have been completed (Lopez-Berestein etal., J. Infect. Dis., 151(4):704-710, 1985a; 1985b; Coune, Infection,16(3):141-147, 1988; Sculier et al., Eur. J. Cancer Clin. Oncol.,24(3):527-538, 1988). Furthermore, several studies suggest that the useof liposomes is not associated with autoimmune responses, toxicity orgonadal localization after systemic delivery (Mon & Fukatsu, Epilepsia,33(6):994-1000, 1992).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Liposomes bear resemblance to cellular membranes and are contemplatedfor use in connection with the present invention as carriers for thepeptide compositions. They are widely suitable as both water- andlipid-soluble substances can be entrapped, i.e. in the aqueous spacesand within the bilayer itself, respectively. It is possible that thedrug-bearing liposomes may even be employed for site-specific deliveryof active agents by selectively modifying the liposomal formulation.

In addition to the teachings of Couvreur et al. (1977; 1988), thefollowing information may be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins, such as cytochromec, bind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for antibiotic and inhibitordelivery will contain cholesterol.

The ability to trap solutes varies between different types of liposomes.For example, MLVs are moderately efficient at trapping solutes, but SUVsare extremely inefficient. SUVs offer the advantage of homogeneity andreproducibility in size distribution, however, and a compromise betweensize and trapping efficiency is offered by large unilamellar vesicles(LUVs). These are prepared by ether evaporation and are three to fourtimes more efficient at solute entrapment than MLVs.

In addition to liposome characteristics, an important determinant inentrapping compounds is the physicochemical properties of the compounditself. Polar compounds are trapped in the aqueous spaces and nonpolarcompounds bind to the lipid bilayer of the vesicle. Polar compounds arereleased through permeation or when the bilayer is broken, but nonpolarcompounds remain affiliated with the bilayer unless it is disrupted bytemperature or exposure to lipoproteins. Both types show maximum effluxrates at the phase transition temperature.

Liposomes interact with cells via four different mechanisms: endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

The fate and disposition of intravenously injected liposomes depend ontheir physical properties, such as size, fluidity, and surface charge.They may persist in tissues for h or days, depending on theircomposition, and half lives in the blood range from min to several h.Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehaviour limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow, and lymphoid organs.

Targeting is generally not a limitation in terms of the presentinvention. However, should specific targeting be desired, methods areavailable for this to be accomplished. Antibodies may be used to bind tothe liposome surface and to direct the antibody and its drug contents tospecific antigenic receptors located on a particular cell-type surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

Alternatively, the invention provides for pharmaceutically-acceptablenanocapsule formulations of the compositions of the present invention.Nanocapsules can generally entrap compounds in a stable and reproducibleway (Henry-Michelland et al., Journal of Pharmacy and Pharmacology,39(12):973-977, 1987; Quintanar-Guerrero et al., Drug Dev. Ind. Pharm.24(12):1113-1128, 1998; Douglas et al., Crit. Rev. Ther. Drug CarrierSyst., 3(3):233-261, 1987). To avoid side effects due to intracellularpolymeric overloading, such ultrafine particles (sized around 0.1 μm)should be designed using polymers able to be degraded in vivo.Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet theserequirements are contemplated for use in the present invention. Suchparticles may be are easily made, as described (Couvreur et al., J.Pharm. Sci., 69(2):199-202, 1980; 1988; zur Muhlen et al., Eur. J.Pharm. Biopharm., 45(2):149-155, 1998; Zambaux et al., J. Control.Release. 50(1-3):31-40, 1998; Pinto-Alphandry et al., J. Drug Target.,3:167-169, 1995 and U.S. Pat. No. 5,145,684, specifically incorporatedherein by reference in its entirety).

Skin patches may also be utilised for transcutaneous delivery.

Immunogenic Compositions

In certain preferred embodiments of the present invention, immunogeniccompositions are provided. The immunogenic compositions will generallycomprise one or more polypeptides or polynucleotides, such as thosediscussed above, in combination with an immunostimulant. Animmunostimulant may be any substance that enhances or potentiates animmune response (antibody and/or cell-mediated) to an exogenous antigen.Examples of immunostimulants include adjuvants, biodegradablemicrospheres (e.g., polylactic galactide) and liposomes (into which thecompound is incorporated; see, e.g., Fullerton, U.S. Pat. No.4,235,877).

Preparation of immunogenic compositions is generally described in, forexample, Powell & Newman, eds., Vaccine Design (the subunit and adjuvantapproach) (1995). Pharmaceutical compositions and immunogeniccompositions within the scope of the present invention may also containother compounds, which may be biologically active or inactive. Forexample, one or more immunogenic portions of other M. tuberculosisantigens may be present, either incorporated into a fusion polypeptideor as a separate compound, within the pharmaceutical or immunogeniccomposition.

Illustrative immunogenic compositions may contain a polynucleotide (e.g.DNA) encoding one or more of the polypeptides as described above, suchthat the polypeptide is generated in situ (thereby eliciting an immuneresponse). As noted above, the DNA may be present within any of avariety of delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacteria and viral expressionsystems. Numerous gene delivery techniques are well known in the art,such as those described by Rolland, Crit. Rev. Therap. Drug CarrierSystems 15:143-198 (1998), and references cited therein. Appropriatenucleic acid expression systems contain the necessary DNA sequences forexpression in the patient (such as a suitable promoter and terminatingsignal). Bacterial delivery systems involve the administration of abacterium host cell (for example, a Mycobacterium, Bacillus orLactobacillus strain, including Bacillus-Calmette-Guerrin or Lactococcuslactis) that expresses the polypeptide (e.g. on its cell surface orsecretes the polypeptide) (see, for example, Ferreira, et al., An AcadBras Cienc (2005) 77:113-124; and Raha, et al., Appl MicrobiolBiotechnol (2005) PubMedID 15635459). In a preferred embodiment, the DNAmay be introduced using a viral expression system (e.g., vaccinia orother pox virus, retrovirus, or adenovirus), which may involve the useof a non-pathogenic (defective), replication competent virus. Suitablesystems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl.Acad. Sci. USA 86:317-321 (1989); Flexner et al., Ann. N.Y. Acad. Sci.569:86-103 (1989); Flexner et al., Vaccine 8:17-21 (1990); U.S. Pat.Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627 (1988); Rosenfeld et al., Science 252:431-434(1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994);Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502 (1993);Guzman et al., Circulation 88:2838-2848 (1993); and Guzman et al., Cir.Res. 73:1202-1207 (1993). Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in Ulmer et al.,Science 259:1745-1749 (1993) and reviewed by Cohen, Science259:1691-1692 (1993). The uptake of naked DNA may be increased bycoating the DNA onto biodegradable beads, which are efficientlytransported into the cells. It will be apparent that a immunogeniccomposition may comprise both a polynucleotide and a polypeptidecomponent. Such immunogenic composition may provide for an enhancedimmune response.

It will be apparent that an immunogenic composition may containpharmaceutically acceptable salts of the polynucleotides andpolypeptides provided herein. Such salts may be prepared frompharmaceutically acceptable non-toxic bases, including organic bases(e.g., salts of primary, secondary and tertiary amines and basic aminoacids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium,calcium and magnesium salts).

While any suitable carrier known to those of ordinary skill in the artmay be employed in the immunogenic compositions of this invention, thetype of carrier will vary depending on the mode of administration.Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;5,814,344 and 5,942,252. One may also employ a carrier comprising theparticulate-protein complexes described in U.S. Pat. No. 5,928,647,which are capable of inducing a class I-restricted cytotoxic Tlymphocyte responses in a host.

Such compositions may also comprise buffers (e.g., neutral bufferedsaline or phosphate buffered saline), carbohydrates (e.g., glucose,mannose, sucrose or dextrans), mannitol, proteins, polypeptides or aminoacids such as glycine, antioxidants, bacteriostats, chelating agentssuch as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide),solutes that render the formulation isotonic, hypotonic or weaklyhypertonic with the blood of a recipient, suspending agents, thickeningagents and/or preservatives. Alternatively, compositions of the presentinvention may be formulated as a lyophilizate. Compounds may also beencapsulated within liposomes using well known technology.

Any of a variety of immunostimulants may be employed in the immunogeniccompositions of this invention. For example, an adjuvant may beincluded. Most adjuvants contain a substance designed to protect theantigen from rapid catabolism, such as aluminium hydroxide or mineraloil, and a stimulator of immune responses, such as lipid A, Bortadellapertussis or Mycobacterium species or Mycobacterium derived proteins.For example, delipidated, deglycolipidated M. vaccae (“pVac”) can beused. Suitable adjuvants are commercially available as, for example,Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories,Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway,N.J.); AS01B, AS02A, AS15, AS-2 and derivatives thereof(GlaxoSmithKline, Philadelphia, Pa.); CWS (cell wall skeleton from atubercule bacillus), TDM (trehalose dicorynomycolate), Leif (Leishmaniaelongation initiation factor), aluminium salts such as aluminiumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A(MPL®); and quil A (e.g. QS-21). Cytokines, such as GM-CSF orinterleukin-2, -7, or -12, may also be used as adjuvants.

An adjuvant refers to the components in a vaccine or therapeuticcomposition that increase the specific immune response to the antigen(see, e.g., Edelman, AIDS Res. Hum Retroviruses 8:1409-1411 (1992)).Adjuvants induce immune responses of the Th1-type and Th-2 typeresponse. Th1-type cytokines (e.g., IFN-γ, IL-2, and IL-12) tend tofavour the induction of cell-mediated immune response to an administeredantigen, while Th-2 type cytokines (e.g., IL-4, IL-5, 11-6, IL-10) tendto favour the induction of humoral immune responses. Adjuvants capableof preferential stimulation of a Th-1 cell-mediated immune response aredescribed in WO 94/00153 and WO 95/17209.

Within the immunogenic compositions provided herein, the adjuvantcomposition is preferably designed to induce an immune responsepredominantly of the Th1 type. Following application of a immunogeniccomposition as provided herein, a patient will typically support animmune response that includes Th1- and Th2-type responses. Within apreferred embodiment, in which a response is predominantly Th1-type, thelevel of Th1-type cytokines will increase to a greater extent than thelevel of Th2-type cytokines. The levels of these cytokines may bereadily assessed using standard assays. For a review of the families ofcytokines, see Janeway, et al., Immunobiology, 5^(th) Edition, 2001.

The Rv2386c compositions usually comprise one or more adjuvants, e.g.,AS01B (3-de-O-acylated monophosphoryl lipid A (3D-MPL®) and QS21 in aliposome formulation; see, U.S. Patent Publication No. 2003/0143240);AS02A (3D-MPL® and QS21 and an oil in water emulsion; see, Bojang, etal., Lancet (2001) 358:1927); ENHANZYN® (Detox); 3D-MPL®; saponinsincluding Quil A and its components e.g. QS21 and saponin mimetics; CWS(cell wall skeleton from a tubercule bacillus); TDM (trehalosedicorynomycolate); aminoalkyl glucosaminide 4-phosphates (AGPs);immunostimulatory oligonucleoptides e.g. CPG; Leif (Leishmaniaelongation initiation factor); and derivatives thereof. In a preferredembodiment, an Rv2386c polypeptide is administered with one or moreadjuvants selected from the group consisting of 3D-MPL® and QS21 in aliposome formulation eg AS01B and 3D-MPL® and QS21 and an oil in wateremulsion (e.g. AS02A). Adjuvant systems AS01B and AS02A are furtherdescribed in Pichyangkul, et al., Vaccine (2004) 22:3831-40.

When delivering the Rv2386c antigen as a nucleic acid, it can bedelivered, for example, in a viral vector (i.e., an adenovirus vector),or in a mutant bacterium host cell (i.e., a mutant, avirulentMycobacterium, Lactobacillus or Bacillus host cell including BacillusCalmette-Guerin (BCG) and Lactococcus lactis).

Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A(MPL®), preferably 3-O-deacylated monophosphoryl lipid A (3D-MPL®),optionally with an aluminium salt (see, for example, Ribi, et al., 1986,Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ.Corp., NY, pp. 407-419; GB 2122204B; GB 2220211; and U.S. Pat. No.4,912,094). A preferred form of 3D-MPL® is in the form of an emulsionhaving a small particle size less than 0.2 mm in diameter, and itsmethod of manufacture is disclosed in WO 94/21292. Aqueous formulationscomprising monophosphoryl lipid A and a surfactant have been describedin WO 98/43670. Exemplified preferred adjuvants include AS01B (MPL® andQS21 in a liposome formulation), 3D-MPL® and QS21 in a liposomeformulation, AS02A (MPL® and QS21 and an oil in water emulsion), 3D-MPL®and QS21 and an oil in water emulsion, and AS15, available fromGlaxoSmithKline. MPL® adjuvants are available from GlaxoSmithKline (seeU.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094).

CpG-containing oligonucleotides (in which the CpG dinucleotide isunmethylated) also induce a predominantly Th1 response. CpG is anabbreviation for cytosine-guanosine dinucleotide motifs present in DNA.Such oligonucleotides are well known and are described, for example, inWO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462.Immunostimulatory DNA sequences are also described, for example, by Satoet al., Science 273:352 (1996). CpG when formulated into immunogeniccompositions, is generally administered in free solution together withfree antigen (WO 96/02555; McCluskie and Davis, supra) or covalentlyconjugated to an antigen (WO 98/16247), or formulated with a carriersuch as aluminium hydroxide ((Hepatitis surface antigen) Davis et al.supra; Brazolot-Millan et al., Proc. Natl. Acad. Sci., USA, 1998,95(26), 15553-8). CpG is known in the art as being an adjuvant that canbe administered by both systemic and mucosal routes (WO 96/02555, EP468520, Davis et al., J. Immunol, 1998, 160(2):870-876; McCluskie andDavis, J. Immunol., 1998, 161(9):4463-6).

Another preferred adjuvant is a saponin or saponin mimetics orderivatives, such as Quil A, preferably QS21 (Aquila BiopharmaceuticalsInc., Framingham, Mass.), which may be used alone or in combination withother adjuvants. For example, an enhanced system involves thecombination of a monophosphoryl lipid A (MPL®) and saponin derivative,such as the combination of QS21 and 3D-MPL® as described in WO 94/00153,or a less reactogenic composition where the QS21 is quenched withcholesterol, as described in WO 96/33739. Other preferred formulationscomprise an oil-in-water emulsion and tocopherol. A particularly potentadjuvant formulation involving QS21, 3D-MPL® and tocopherol in anoil-in-water emulsion is described in WO 95/17210. Additional saponinadjuvants of use in the present invention include QS7 (described in WO96/33739 and WO 96/11711) and QS17 (described in U.S. Pat. No. 5,057,540and EP 0 362 279 B1).

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOM®s. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM®. Thesaponins may also be formulated with excipients such as CARBOPOL® toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

In one embodiment, the adjuvant system includes the combination of amonophosphoryl lipid A and a saponin derivative, such as the combinationof QS21 and 3D-MPL® adjuvant, as described in WO 94/00153, or a lessreactogenic composition where the QS21 is quenched with cholesterolcontaining liposomes, as described in WO 96/33739. Other suitableformulations comprise an oil-in-water emulsion and tocopherol. Anothersuitable adjuvant formulation employing QS21, 3D-MPL® adjuvant andtocopherol in an oil-in-water emulsion is described in WO 95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 as disclosed in WO 00/09159. Suitably theformulation additionally comprises an oil in water emulsion andtocopherol.

Other suitable adjuvants include MONTANIDE® ISA 720 (Seppic, France),SAF (Chiron, Calif., United States), ISCOMS® (CSL), MF-59 (Chiron), theSBAS series of adjuvants (SmithKline Beecham, Rixensart, Belgium), Detox(Corixa), RC-529 (Corixa) and other aminoalkyl glucosaminide4-phosphates (AGPs), such as those described in pending U.S. patentapplication Ser. Nos. 08/853,826 and 09/074,720 (U.S. Pat. Nos.6,113,918 and 6,355,257, respectively), the disclosures of which areincorporated herein by reference in their entireties, andpolyoxyethylene ether adjuvants such as those described in WO99/52549A1. SmithKline Beecham and Corixa Corporation are now part ofGlaxoSmithKline.

Other suitable adjuvants include adjuvant molecules of the generalformula (I):HO(CH₂CH₂O)_(n)-A-Rwherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or PhenylC₁₋₅₀ alkyl.

A further adjuvant of interest is shiga toxin b chain, used for exampleas described in WO2005/112991.

One embodiment of the present invention consists of an immunogeniccomposition comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

Any immunogenic composition provided herein may be prepared using wellknown methods that result in a combination of antigen, immune responseenhancer and a suitable carrier or excipient. The compositions describedherein may be administered as part of a sustained release formulation(i.e., a formulation such as a capsule, sponge or gel (composed ofpolysaccharides, for example) that effects a slow release of compoundfollowing administration). Such formulations may generally be preparedusing well known technology (see, e.g., Coombes et al., Vaccine14:1429-1438 (1996)) and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.

Carriers for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of active component release. Such carriers includemicroparticles of poly(lactide-co-glycolide), polyacrylate, latex,starch, cellulose, dextran and the like. Other delayed-release carriersinclude supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see, e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638). The amount of active compound contained within a sustainedrelease formulation depends upon the site of implantation, the rate andexpected duration of release.

Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and immunogenic compositions to facilitateproduction of an antigen-specific immune response. Delivery vehiclesinclude antigen presenting cells (APCs), such as dendritic cells,macrophages, B cells, monocytes and other cells that may be engineeredto be efficient APCs. Such cells may, but need not, be geneticallymodified to increase the capacity for presenting the antigen, to improveactivation and/or maintenance of the T cell response and/or to beimmunologically compatible with the receiver (i.e., matched HLAhaplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, and may be autologous, allogeneic,syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau& Steinman, Nature 392:245-251(1998)) and have been shown to be effective as a physiological adjuvantfor eliciting prophylactic or therapeutic immunity (see Timmerman &Levy, Ann. Rev. Med. 50:507-529 (1999)). In general, dendritic cells maybe identified based on their typical shape (stellate in situ, withmarked cytoplasmic processes (dendrites) visible in vitro), theirability to take up, process and present antigens with high efficiencyand their ability to activate naïve T cell responses. Dendritic cellsmay, of course, be engineered to express specific cell-surface receptorsor ligands that are not commonly found on dendritic cells in vivo or exvivo, and such modified dendritic cells are contemplated by the presentinvention. As an alternative to dendritic cells, secreted vesiclesantigen-loaded dendritic cells (called exosomes) may be used within animmunogenic composition (see Zitvogel et al., Nature Med. 4:594-600(1998)).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, lymph nodes, spleen, skin, umbilical cord blood or anyother suitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorised as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterised phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterised as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1 BB).

APCs may generally be transfected with a polynucleotide encoding aprotein (or portion or other variant thereof) such that the polypeptide,is expressed on the cell surface. Such transfection may take place exvivo, and a pharmaceutical composition or immunogenic compositioncomprising such transfected cells may then be used, as described herein.Alternatively, a gene delivery vehicle that targets a dendritic or otherantigen presenting cell may be administered to a patient, resulting intransfection that occurs in vivo. In vivo and ex vivo transfection ofdendritic cells, for example, may generally be performed using anymethods known in the art, such as those described in WO 97/24447, or thegene gun approach described by Mahvi et al., Immunology and Cell Biology75:456-460 (1997). Antigen loading of dendritic cells may be achieved byincubating dendritic cells or progenitor cells with the polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

Immunogenic compositions and pharmaceutical compositions may bepresented in unit-dose or multi-dose containers, such as sealed ampoulesor vials. Such containers are preferably hermetically sealed to preservesterility of the formulation until use. In general, formulations may bestored as suspensions, solutions or emulsions in oily or aqueousvehicles. Alternatively, an immunogenic composition or pharmaceuticalcomposition may be stored in a freeze-dried condition requiring only theaddition of a sterile liquid carrier immediately prior to use.

In some embodiments, a “priming” or first administration of an Rv2386cpolypeptide (including variants, immunogenic fragments or fusionproteins), or polynucleotide encoding said polypeptide, is followed byone or more “boosting” or subsequent administrations of an Rv2386cpolypeptide (including variants, immunogenic fragments or fusionproteins) or polynucleotide encoding said polypeptide (“prime and boost”method). For instance, a first administration with an Rv2386cpolypeptide (including variants, immunogenic fragments or fusionproteins) or polynucleotide encoding said polypeptide is followed by oneor more subsequent administrations of an Rv2386c polypeptide (includingvariants, immunogenic fragments or fusion proteins) or polynucleotideencoding said polypeptide.

In one embodiment, a first administration with an Rv2386c polypeptide orpolynucleotide is followed by one or more subsequent administrations ofan Rv2386c polypeptide. In one embodiment, a first administration withan Rv2386c polypeptide or polynucleotide is followed by one or moresubsequent administrations of an Rv2386c polynucleotide. Usually thefirst or “priming” administration and the second or “boosting”administration are given about 2-12 weeks apart, or up to 4-6 monthsapart. Subsequent “booster” administrations are given about 6 monthsapart, or as long as 1, 2, 3, 4 or 5 years apart. Conventional boostertreatment (e.g., a protein priming administration followed by a proteinboosting administration) may also useful be in preventing or treatingtuberculosis (e.g. preventing or treating latent tuberculosis, inparticular preventing or delay tuberculosis reactivation).

Antibodies

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognises an antigen. The recognised immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterised fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesised de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanised antibodies. Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)).

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to fusion proteins can be selected to obtain only thosepolyclonal antibodies that are specifically immunoreactive with fusionprotein and not with individual components of the fusion proteins. Thisselection may be achieved by subtracting out antibodies that cross-reactwith the individual antigens. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) and UsingAntibodies: A Laboratory Manual (1998), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity). Typically a specific or selective reaction will be atleast twice background signal or noise and more typically more than 10,20 or 100 times background (e.g. binding to other Mycobacteriumproteins, such as other Mycobacterium tuberculosis proteins).

Diagnostics

In another aspect, this invention provides methods for using one or moreof the polypeptides described above to diagnose tuberculosis (forexample using T cell response based assays or antibody based assays ofconventional format).

For example, there is provided a method for determining prior M.tuberculosis infection in an individual comprising:

-   -   (a) obtaining a sample from the individual;    -   (b) contacting said sample with an isolated polypeptide which        comprises:        -   (i) an Rv2386c protein sequence;        -   (ii) a variant of an Rv2386c protein sequence; or        -   (iii) an immunogenic fragment of an Rv2386c protein            sequence;    -   (c) quantifying the sample response.

The sample may for example be whole blood or purified cells. Suitablythe sample will contain peripheral blood mononucleated cells (PBMC). Inone embodiment of the invention the individual will be seropositive. Ina second embodiment of the invention the individual will beseronegative.

Suitably the individual will not previously have been vaccinated againstM. tuberculosis infection (e.g. suitably the individual will notpreviously have been vaccinated with BCG).

The sample response may be quantified by a range of means known to thoseskilled in the art, including the monitoring of lymphocyte proliferationor the production of specific cytokines or antibodies. For example,T-cell ELISPOT may be used to monitor cytokines such as interferon gamma(IFNγ), interleukin 2 (IL2) and interleukin 5 (IL5). B-cell ELLISPOT maybe used to monitor the stimulation of M. tuberculosis specific antigens.The cellular response may also be characterised by the use of by intra-and extra-cellular staining and analysis by a flow cytometer.

Methods of quantifying a sample proliferation response include:

-   -   (i) pulsing cultured cells with a radiolabel (e.g. tritiated        thymidine) and monitoring tritium uptake (e.g. gas        scintillation);    -   (ii) carboxyfluorsecein diacetate succinimidyl ester (CFSE)        labelling and fluorescence monitoring of cell division using        flow cytometry.

Quantifying a sample cytokine response includes in particular themonitoring of interferon gamma production.

When using such quantification methods, a positive response to anantigen may be defined by a signal to noise ratio (S/N ratio) of atleast 2:1 (for example, at least 3:1 or at least 5:1).

In a further aspect of the present invention methods are provided todiagnose M. tuberculosis infection using a skin test. As used herein, a“skin test” is any assay performed directly on a patient in which adelayed-type hypersensitivity (DTH) reaction (such as swelling,reddening or dermatitis) is measured following intradermal injection ofan Rv153c polypeptide as described above (or variant, immunogenicfragments thereof or nucleotides encoding them). Such injection may beachieved using any suitable device sufficient to contact the antigencombinations with dermal cells of the patient, such as a tuberculinsyringe or 1 mL syringe. The reaction is measured after a period oftime, for example at least 48 hours after injection, especially 48-72hours.

The DTH reaction is a cell-mediated immune response, which is greater inpatients that have been exposed previously to the test antigen. Theresponse may be measured visually, using a ruler. In general, a responsethat is greater than about 0.5 cm in diameter, especially greater thanabout 1.0 cm in diameter, is a positive response, indicative of prior M.tuberculosis infection, which may or may not be manifested as an activedisease.

For use in a skin test, the Rv2386c component is suitably formulated asa pharmaceutical composition containing a physiologically acceptablecarrier. Suitably, the carrier employed in such pharmaceuticalcompositions is a saline solution with appropriate preservatives, suchas phenol and/or Tween™ 80.

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment.

For example, one container within a kit may contain a monoclonalantibody or fragment thereof that specifically binds to a protein. Suchantibodies or fragments may be provided attached to a support material,as described above. One or more additional containers may encloseelements, such as reagents or buffers, to be used in the assay. Suchkits may also, or alternatively, contain a detection reagent asdescribed above that contains a reporter group suitable for direct orindirect detection of antibody binding.

Alternatively, a kit may be designed to detect the level of mRNAencoding a protein in a biological sample. Such kits generally compriseat least one oligonucleotide probe or primer, as described above, thathybridises to a polynucleotide encoding a protein. Such anoligonucleotide may be used, for example, within a PCR or hybridizationassay. Additional components that may be present within such kitsinclude a second oligonucleotide and/or a diagnostic reagent orcontainer to facilitate the detection of a polynucleotide encoding aprotein of the invention.

Other diagnostics kits include those designed for the detection of cellmediated responses (which may, for example, be of use in the diagnosticmethods of the present invention). Such kits will typically comprise:

-   -   (i) apparatus for obtaining an appropriate cell sample from a        subject;    -   (ii) means for stimulating said cell sample with an Rv2386c        polypeptide (or variant thereof, immunogenic fragments thereof,        or DNA encoding such polypeptides);    -   (iii) means for detecting or quantifying the cellular response        to stimulation.

Suitable means for quantifying the cellular response include a B-cellELISPOT kit or alternatively a T-cell ELISPOT kit, which are known tothose skilled in the art.

One possible kit comprises:

-   -   (a) a polypeptide of the invention; and    -   (b) a detection reagent suitable for direct or indirect        detection of antibody binding.

Of particular interest are diagnostic kits tailored for quantifying Tcell responses:

-   -   A diagnostic kit comprising:    -   (a) a polypeptide of the invention; and    -   (b) apparatus sufficient to contact said polypeptide with the        dermal cells of an individual.    -   A diagnostic kit comprising:    -   (a) a polypeptide of the invention;    -   (b) apparatus sufficient to contact said polypeptide with a        sample (e.g. whole blood or more suitably PBMC) from an        individual; and    -   (c) means to quantify the T cell response (e.g. proliferation or        IFN-gamma production).

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example 1 Identification of Rv2386c as a Latent TB Vaccine Target

The gene Rv2386c also known as Mbtl, encodes for a protein which isinvolved in the biogenesis of hydroxyphenyloxazoline-containingsiderophore mycobactins where it converts chorismate to salicylate, thestarter unit for mycobacterin siderophore synthesis.

Rv2386c was selected based on a genome-wide analysis of Mycobacteriumtuberculosis genes associated with dormancy phase maintenance andinfectivity as in Murphy and Brown BMC. Infect. Dis. 2007 7:84-99.Potential dormancy phase gene targets in Mycobacterium tuberculosis wereprioritised through a bioinformatics meta-analysis of publishedgenome-wide DNA microarray datasets of bacterial gene expression undersimulated dormancy conditions. Subcellular localisation of M.tuberculosis proteins encoded by genes, was subsequently carried out onthe entire genome to identify vaccine targets.

Briefly, experimental conditions in the dormancy models were quitevaried so a zero to five scoring system was developed to normalise thesedata based upon two criteria: 1) the relevance of the experimentalconditions to the dormant state and 2) the rank order of expression. Themaximum score for a particular experimental dataset was adjusted basedon potential relevance to the clinical occurrence of dormancy phase M.tuberculosis infections. Table 1 shows the data sets collected for Step1 together with the adjusted maximum scores for each dataset. Additionaldatasets on gene essentiality for growth were obtained from publishedstudies using transposon-based knockout experiments (TraSH). Genes whichhad no effect on growth received a score of zero.

TABLE 1 Sources, experimental models, and scoring criteria for M.tuberculosis DNA microarray gene expression and genome-wide geneknock-out (growth phase essentiality). Timepoint: Reference Experimentalmodel Maximum score^(a) Betts J C et al. Starvation under controlled O₂96 h: 3 Mol. Microbiol. 2002 43: 717-731 24 h: 2 4 h: 1 Hampshire T etal. Nutrient depletion under 62 and 75 d: 5 Tuberculosis. (Edinb.) 200484: 228-238 controlled O₂ 49 d: 4 18 d: 2 Muttucumaru D G et al. Waynemodel of hypoxia^(#) 14 d (NRP-2): 4 Tuberculosis. (Edinb.) 2004 84:239-246 7 d (NRP-1): 2 Voskuil M I et al. Wayne model of hypoxia^(#) 30and 80 d: 5 Tuberculosis. (Edinb.) 2004 84: 218-227 14 and 20 d: 4 10and 12 d: 3 6 and 8 d: 2 Schnappinger D et al. Infection of mousemacrophages, 24 and 48 h: 5 J. Exp. Med. 2003 198: 693-704 +/−γ-INFKarakousis P C et al. Hollow fiber subcutaneous 10 d: 3 J. Exp. Med.2004 200: 647-657 implant in mice Talaat A M et al. Infection of mice.MTB harvested 28 d: 3 Proc. Natl. Acad. Sci. U.S.A 2004, from lung^(b)101: 4602-4607 Sassetti C M et al. TraSH mutated libraries grown on 14d: 5 Mol. Microbiol. 2003 48: 77-84 solid media Rengarajan J et al.Infection of mouse macrophages, 7 d: 5 Proc. Natl. Acad. Sci. U.S.A2005, +/−γ-INF with TraSH mutated 102: 8327-8332 libraries of M.tuberculosis Sassetti C M et al. C57BL/6J mice infected with 7, 14, 28and 56 d: 5 Proc. Natl. Acad. Sci. U.S.A 2003 TraSH mutated libraries ofM. tuberculosis 100: 12989-12994 ^(a)Maximum score based on relevance asa dormancy model; h = hour; d = day. ^(b)Ratio of M. tuberculosis fromBalb/c lung to MTB in aerated culture for 28 d. ^(#)Wayne L G and HayesL G Infect. Immun. 1996 64: 2062-2069

Step 2—In applying the second criterion, the rank order of geneexpression, gene scores from each dataset were ordered from highest tolowest based on expression ratio (fold expression in the experimentalcondition versus cells in log-phase liquid culture). The highest scoringgene received the maximum score for that particular dataset (listed incolumn 3 of Table 1. (e.g. 5, 4 . . . , 1 point)). The score wasdecreased by 0.005 points for each gene in order until zero, or the endof the data set was reached. Thus when the maximum score was 4 points,the 100th ranked gene would receive a score of 3.500. For a maximumscore of 5 points, 1000 genes or 25% of the M. tuberculosis genomereceived a score. For experiments where data from multiple time pointswere collected, the maximum score across all time points was used as thefinal score.

In Step 3 scores for each gene in each of the experimental conditionswere collected into a Microsoft Access database. Reference fields wereadded to facilitate prioritisation, such as the Refseq ID, Genbankfunction, Genbank note, Tuberculist classification, and KEGG and SangerCenter links. By combining the data from different studies and sources,a consensus view was reached about the particular genes and pathwaysmost critical for survival in the dormant state.

In Step 4, a prioritised list of therapeutic targets was derivedutilising the top 400 scoring genes (˜10% of the genome) supplemented byexpert computational and manual analysis of biochemical pathways,enzymology, drug tractability, homology to human genes and other priorknowledge. The great majority of the high scoring genes come from thesubset where two or three of the groups intersect.

In Step 5, the identification of subcellular localisation of M.tuberculosis proteins encoded by genes, was carried out on the entiregenome. The heuristic used for membrane protein prediction is describedin Chalker et al. J. Bacteriol. 2001 183:1259-1268. Average hydropathyprofiles (H) (von Heijne G J. Mol. Biol. 1992 225:487-494) weregenerated using GES hydropathy values (Engelman D M et al. Annu. Rev.Biophys. Biophys. Chem. 1986 15:321-353) weighted using a trapezoidwindow. Using a process similar to the initial steps of the TopPred IIalgorithm (Claros M G et al. Comput. Appl. Biosci. 1994 10:685-686),helical transmembrane segments (TMS) were predicted for each peptidesequence by selecting 19 amino acids centered on the highest H value(MaxH), masking these from further consideration, and repeating theprocess until no peaks with a H of >0.5 remained. Subcellular locationswere assigned based on the peak MaxH value, number of segments with a Hof >1.0, and distribution and peak H values of the putative TMS. A MaxHcutoff of 1.15 was chosen to maximize the discrimination between twoSwissProtein release 34 test datasets containing transmembrane andcytoplasmic proteins, respectively (Boyd D et al. Protein Sci. 19987:201-205). Proteins with a MaxH of <1.15 were classified ascytoplasmic, while those with a MaxH of >1.15 and at least threepossible TMS were classified as membrane proteins. Anchored proteinswere defined as having exactly two TMS, one starting before amino acid(aa) 35 and one having a H of >1.15 with the other having a H not lowerthan 0.5. SignalP with Gram positive settings was specifically used forM. bacterium to identify secreted proteins amongst those classified aseither cytoplasmic or “unknown” in the heuristic analysis (Nielsen H etal. Protein Eng. 1997 10:1-6).

Rv2386c ranked very high as a vaccine antigen according to severalcriteria:

-   -   (i) Rv2386c is consistently up-regulated across all models of        dormancy. Among the entire suite of 3999 genes scored in the        meta-analysis, Rv2386c was ranked 120^(th) as one of the top 10%        of over-expressed genes across all dormancy models. The        up-regulated score for Rv2386c was 13.165 which favourably        compared with the top gene score of 22.28. Rv2386c was minimally        down-regulated, scoring only slightly less than 0 (−2.316        compared to −18.13 for the most widely down regulated gene).    -   (ii) The scavenging of iron from macrophages is crucial for M.        tuberculosis survival and infectivity. Small molecules called        siderophores function in bacteria to bind iron for        intra-cellular uptake, Mbtl catalyses the first step in the        formation of the only M. tuberculosis siderophore, mycobactin T,        the biosynthesis of which depends on the production of        salicylate from chorismate (Harrison et al. J. Bacteriol. 2006        188:6081-6091). Expression of Mbtl is induced during M.        tuberculosis infection of human THP-1 macrophages (Gold et al.        Mol. Microbiol. 2001 42:851-865). The 3D protein structure is        available and the mechanism of enzymatic action Mbtl has been        well-studied (Zwahlen et al. Biochemistry 2007 46:954-964).        Rv2386c ranked high in essentiality scoring and is required        by M. tuberculosis for survival in in vitro growth models        (scoring 2.6 out of a possible scoring of 5).    -   (iii) Subcellular localisation predicted that the Rv2386c        protein is secreted and thus has significant extracellular        exposure, indicating suitability as a vaccine target.

Example 2 Rv2386c Epitope Identification

Method

T cell epitope prediction was based on the following approaches:

Prediction Name URL/References CD4 and Multipred website:antigen.i2r.a-star.edu.sg/multipred/ CD8 Zhang, G. L., Khan, A. M.,Srinivasan, K. N., August, J. T. and Brusic, V. (2005) “MULTIPRED: acomputational system for prediction of promiscuous HLA binding peptides”Nucleic Acids Res. 33, W172-W179. SVMHC website:www-bs.informatik.uni-tuebingen.de/SVMHC “Prediction of MHC class Ibinding peptides, using SVMHC.” Pierre Dönnes and Arne Elofsson in: BMCBioinformatics 2002 3: 25 CD4 ProPred website:www.imtech.res.in/raghava/propred/ Singh, H. and Raghava, G. P. S.(2001) “ProPred: Prediction of HLA-DR binding sites.” Bioinformatics,17(12), 1236-37. Tepitope2 In house program based on: H. Bian, J. Hammer(2004) “Discovery of promiscuous HLA-II- restricted T cell epitopes withTEPITOPE.” Methods 34: 468-75 CD8 nHLA website:www.imtech.res.in/raghava/nhlapred/ Bhasin M. and Raghava G P S (2006)“A hybrid approach for predicting promiscuous MHC class I restricted Tcell epitopes”; J. Biosci. 32: 31-42 NetCTL website:www.cbs.dtu.dk/services/NetCTL/ “An integrative approach to CTL epitopeprediction. A combined algorithm integrating MHC-I binding, TAPtransport efficiency, and proteasomal cleavage predictions.” Larsen M.V., Lundegaard C., Kasper Lamberth, Buus S,. Brunak S., Lund O., andNielsen M. European Journal of Immunology. 35(8): 2295-303. 2005 Epijenwebsite: www.jenner.ac.uk/EpiJen/ Doytchinova, I. A., P. Guan, D. R.Flower. “EpiJen: a server for multi-step T cell epitope prediction.” BMCBioinformatics, 2006, 7, 131. Syfpeithi website:www.syfpeithi.de/Scripts/MHCServer.dll/EpitopePrediction.htm Hans-GeorgRammensee, Jutta Bachmann, Niels Nikolaus Emmerich, Oskar AlexanderBachor, Stefan Stevanovic: “SYFPEITHI: database for MHC ligands andpeptide motifs.” Immunogenetics (1999) 50: 213-219 PredTAP website:antigen.i2r.a-star.edu.sg/predTAP/ Zhang, G. L., Petrovsky, N., Kwoh, C.K., August, J. T. and Brusic, V. (2006) “M PRED^(TAP): a system forprediction of peptide binding to the human transporter associated withantigen processing.” Immunome Res. 2(1), 3. PAPROC website:www.paproc2.de/paproc1/paproc1.html C. Kuttler, A. K. Nussbaum, T. P.Dick, H.-G. Rammensee, H. Schild, K. P. Hadeler, “An algorithm for theprediction of proteasomal cleavages”, J. Mol. Biol. 298 (2000), 417-429A. K. Nussbaum, C. Kuttler, K. P. Hadeler, H.-G. Rammensee, H. Schild,“PAProC: A Prediction Algorithm for Proteasomal Cleavages available onthe WWW”, Immunogenetics 53 (2001), 87-94Results

TABLE 2 Putative Rv2386c human CD4+ T cell epitopes Putative CD4 Aminoepitope acid Epitope SEQ ID HLA number position sequence No: allele 1 54WVLAAGVQA SEQ ID DRB1_0401 No: 30 2 55 VLAAGVQAM SEQ ID DRB1_0301,No: 31 DRB1_1301 3 73 VIRDGVTRR SEQ ID DRB1_0301, No: 32 DRB1_0401 4 125LAPHTPLAR SEQ ID DRB1_1301 No: 33 5 148 IRLFDAGIR SEQ ID DRB1_1101,No: 34 DRB1_1301, DRB1_1501 6 155 IRHREAIDR SEQ ID DRB1_0801, No: 35DRB1_1301 7 164 LLATGVREV SEQ ID DRB1_1301 No: 36 8 187 FRRRVAVAV SEQ IDDRB1_0101, No: 37 DRB1_0801 9 216 FAIDFPLTY SEQ ID DRB1_0301, No: 38DRB1_0401 10 220 FPLTYRLGR SEQ ID DRB1_1101 No: 39 11 224 YRLGRRHNTSEQ ID DRB1_0801, No: 40 DRB1_1101, DRB1_1501 12 234 VRSFLLQLG SEQ IDDRB1_1301 No: 41 13 238 LLQLGGIRA SEQ ID DRB1_1501 No: 42 14 253LVTAVRADG SEQ ID DRB1_1301 No: 43 15 257 VRADGVVIT SEQ ID DRB1_0301,No: 44 DRB1_0401 16 262 VVITEPLAG SEQ ID DRB1_0301, No: 45 DRB1_0401,DRB1_1301, DRB1_1501 17 295 IVEHAISVR SEQ ID DRB1_1301 No: 46 18 321IDFMTVRER SEQ ID DRB1_1301 No: 47 19 375 FRLDECPRG SEQ ID DRB1_0301,No: 48 DRB1_0401 20 384 LYSGAVVML SEQ ID DRB1_0301 No: 49 21 385YSGAVVMLS SEQ ID DRB1_0401, No: 50 DRB1_1101 22 389 VVMLSADGG SEQ IDDRB1_0401, No: 51 DRB1_1301 23 415 WLRAGAGII SEQ ID DRB1_0101 No: 52

TABLE 3 Putative Rv2386c human CD8+ T cell epitopes Putative CD8 Aminoepitope acid Epitope number position sequence SEQ ID No: HLA allele 1 3ELSVATGAV SEQ ID No: 53 A_0201 2 5 SVATGAVST SEQ ID No: 54 A_0201 3 18IPMPAGVNP SEQ ID No: 55 B_0702, B51, Cw_0401 4 19 PMPAGVNPASEQ ID No: 56 A_0201 5 21 PAGVNPADL SEQ ID No: 57 A_2402, B_3501 6 23GVNPADLAA SEQ ID No: 58 A3 7 25 NPADLAAEL SEQ ID No: 59A24, B_0702, B44,   Cw_0401, Cw_0602, B7, B_3501, B51 8 28 DLAAELAAVSEQ ID No: 60 A_0201 9 33 LAAVVTESV SEQ ID No: 61 A2, A_0201, B51 10 37VTESVDEDY SEQ ID No: 62 A1, A_0101, A_0301 11 38 TESVDEDYL SEQ ID No: 63B44 12 40 SVDEDYLLY SEQ ID No: 64 A1, A_0101, A3 13 48 YECDGQWVLSEQ ID No: 65 A24, B_4403, B51, Cw_0401, B44 14 52 GQWVLAAGVSEQ ID No: 66 A2, A_0201 15 55 VLAAGVQAM SEQ ID No: 67 A2, B8 16 65ELDSDELRV SEQ ID No: 68 A_0201 17 67 DSDELRVIR SEQ ID No: 69A_0101, A_0301 18 82 QQWSGRPGA SEQ ID No: 70 A_0201, A_0301 19 86GRPGAALGE SEQ ID No: 71 A3, B8 20 87 RPGAALGEA SEQ ID No: 72 B7, B_0702,B_3501, B51 21 91 ALGEAVDRL SEQ ID No: 73 A2, A_0201 22 93 GEAVDRLLLSEQ ID No: 74 B44 23 106 AFGWVAFEF SEQ ID No: 75 A24 24 124 RLAPHTPLASEQ ID No: 76 A2, A_0201, A3 25 125 LAPHTPLAR SEQ ID No: 77A_0101, A_0301 26 126 APHTPLARV SEQ ID No: 78 B7, B_3501, B51 27 127PHTPLARVF SEQ ID No: 79 A24, B44 28 130 PLARVFSPR SEQ ID No: 80 A_0101, A3, A_0301 29 133 RVFSPRTRI SEQ ID No: 81  B7 30 134 VFSPRTRIMSEQ ID No: 82  A24, B8 31 143 VSEKEIRLF SEQ ID No: 83  A1, A24 32 153AGIRHREAI SEQ ID No: 84  B8, B51 33 164 LLATGVREV SEQ ID No: 85 A2, A_0201 34 170 REVPQSRSV SEQ ID No: 86  B44 35 172 VPQSRSVDVSEQ ID No: 87  B7, B8 36 180 VSDDPSGFR SEQ ID No: 88  A_0101, A_0301 37183 DPSGFRRRV SEQ ID No: 89  B_3501, B51 38 185 SGFRRRVAV SEQ ID No: 90 B8, B51 39 186 GFRRRVAVA SEQ ID No: 91  A_0301, B8 40 187 FRRRVAVAVSEQ ID No: 92  A3, B8, B51 41 190 RVAVAVDEI SEQ ID No: 93  A24, B7 42198 IAAGRYHKV SEQ ID No: 94  B8, B51 43 200 AGRYHKVIL SEQ ID No: 95B7, B8 44 202 RYHKVILSR SEQ ID No: 96  A3, A24 45 206 VILSRCVEVSEQ ID No: 97  A2, A_0201 46 214 VPFAIDFPL SEQ ID No: 98 B7, B_3501, B51 47 216 FAIDFPLTY SEQ ID No: 99  A1, A_0101, B_3501, B5148 218 IDFPLTYRL SEQ ID No: 100 B44 49 226 LGRRHNTPV SEQ ID No: 101B8, B51 50 228 RRHNTPVRS SEQ ID No: 102 A3, B8 51 231 NTPVRSFLLSEQ ID No: 103 A_0101, A24 52 233 PVRSFLLQL SEQ ID No: 104 A_0201, B7 53245 RALGYSPEL SEQ ID No: 105 A2, A_0201, B_3501, B51 54 246 ALGYSPELVSEQ ID No: 106 A_0101, A2, A_0201 55 249 YSPELVTAV SEQ ID No: 107 A2 56250 SPELVTAVR SEQ ID No: 108 B_0702, B_3501, B51 57 256 AVRADGVVISEQ ID No: 109 A3, A_0301, B7 58 264 ITEPLAGTR SEQ ID No: 110A_0101, A_0301 59 266 EPLAGTRAL SEQ ID No: 111 B7, B8, B_3501, B51 60270 GTRALGRGP SEQ ID No: 112 A3, B8 61 272 RALGRGPAI SEQ ID No: 113A24, B7, B51 62 288 LESNSKEIV SEQ ID No: 114 B44 63 294 EIVEHAISVSEQ ID No: 115 A_0201 64 295 IVEHAISVR SEQ ID No: 116 A_0101 65 298HAISVRSSL SEQ ID No: 117 B7, B8, B_3501 66 301 SVRSSLEEI SEQ ID No: 118B7 67 305 SLEEITDIA SEQ ID No: 119 A2 68 311 DIAEPGSAA SEQ ID No: 120A_0101, A_0301 69 313 AEPGSAAVI SEQ ID No: 121 B44 70 327 RERGSVQHLSEQ ID No: 122 B7, B44 71 331 SVQHLGSTI SEQ ID No: 123 A24, B7 72 342RLDPSSDRM SEQ ID No: 124 A2, A_0201 73 344 DPSSDRMAA SEQ ID No: 125B7, B_3501 74 346 SSDRMAALE SEQ ID No: 126 A1, B8 75 348 DRMAALEALSEQ ID No: 127 B7, Cw_0401, Cw_0602 76 349 RMAALEALF SEQ ID No: 128 A2477 351 AALEALFPA SEQ ID No: 129 A2, A_0201, B_3501 78 352 ALEALFPAVSEQ ID No: 130 A_0101, A2, A_0201 79 353 LEALFPAVT SEQ ID No: 131B8, B_4403 80 357 FPAVTASGI SEQ ID No: 132 B7, B8, B_3501, B51 81 359AVTASGIPK SEQ ID No: 133 A3, A_0301, B7 82 360 VTASGIPKA SEQ ID No: 134A_0201 83 364 GIPKAAGVE SEQ ID No: 135 A_0301, B8 84 365 IPKAAGVEASEQ ID No: 136 B7, B_3501 85 367 KAAGVEAIF SEQ ID No: 137 A24, B_3501 87369 AGVEAIFRL SEQ ID No: 138 A_0201, B51 87 376 RLDECPRGL SEQ ID No: 139A2, A_0201 88 380 CPRGLYSGA SEQ ID No: 140 B7, B_3501 89 383 GLYSGAVVMSEQ ID No: 141 A2, A_0201, A_0301, A3 90 384 LYSGAVVML SEQ ID No: 142A24, B44 91 390 VMLSADGGL SEQ ID No: 143 A2, A_0201, A24 92 397GLDAALTLR SEQ ID No: 144 A_0101, A3, A_0301 93 398 LDAALTLRASEQ ID No: 145 A_0201, B_4403 94 400 AALTLRAAY SEQ ID No: 146A_0101, A_0301, B_3501 95 402 LTLRAAYQV SEQ ID No: 147 A_0201 96 407AYQVGGRTW SEQ ID No: 148 A24 97 414 TWLRAGAGI SEQ ID No: 149 A24 98 424EESEPEREF SEQ ID No: 150 B44 99 430 REFEETCEK SEQ ID No: 151 B44 100 438KLSTLTPYL SEQ ID No: 152 A2, A_0201 101 440 STLTPYLVA SEQ ID No: 153A_0101, A_0201 102 441 TLTPYLVAR SEQ ID No: 154 A3, A_0301

As can be seen from Tables 2 and 3, Rv2386c contains a number ofpredicted CD4+ and CD8 T cell epitopes. Furthermore, this informationsuggests that the protein carries epitopes that can be recognised byHLAs which occur worldwide (that is HLAs from Caucasian, African, Asianor Latin-American individuals available on the worldwide web atallelefrequencies.net).

Example 3 H37Rv Homologues

Rv2386c sequences from a number of M. tuberculosis strains and BCG wereidentified using BLASTP searches of GenBank (H37Rv reference sequenceaccession number YP_177877.1

Strain Accession Number % identity CDC1551 NP_336935.1 100 F11YP_001288342.1 100 Haarlem ZP_02247771.1 100 C ZP_00878160.1 99 BCGCAL72388.1 100

Alignment of the homologue sequences indicates a high level of identity.

Biological Assays

Quantification of T Cell Responses to Rv2386c

Polypeptides may be screened for their ability to activate T-cells(induction of proliferation and/or production of cytokines) inperipheral blood mononuclear cell (PBMC) or in whole blood preparationsfrom infected (such as latently infected) individuals.

Latently infected individuals are usually identified by a skin test thathas a diameter above 10 mm and without symptoms, with no Mtb (M.tuberculosis) positive culture, with a negative sputum negative and withno lesion (as detected by chest X-Ray).

A range of in vitro assays can be used based on PBMC samples or wholeblood: after restimulation in presence of the antigen (orvariant/immunogenic fragment thereof as appropriate) the proliferationof the cells may be determined (as measured by CFSE/flow cytometry) orthe production of cytokines quantified (present in the supernatant ofcultured cells and measured by ELISA, or, after intracellular stainingof CD4 and CD8 T cells and analysis by flow cytometry).

For example, PBMC samples may be obtained from heparinised whole bloodby Ficoll-Hypaque density gradient centrifugation following standardprocedures. The cells may then be washed and cryopreserved in liquidnitrogen until testing (for further details see Lalvani A et al. J.Infect. Dis. 1999 180:1656-1664).

T Cell Proliferation

The specific immune response may be characterised by performinglymphocyte proliferation analysis using the tritiated thymidine. Thistechnique assesses the cellular expansion upon in vitro stimulation toan antigen. In practice, cell proliferation is determined by estimatingincorporation of tritiated-thymidine into DNA, a process closely relatedto underlying changes in cell number.

More suitably, lymphocyte proliferation may be performed using thesuccinimidyl ester of carboxyfluorsecein diacetate (CFSE). CFSEspontaneously and irreversibly couples to both intracellular and cellsurface proteins by reaction with lysine side chains and other availableamine groups. When lymphocyte cells divide, CFSE labelling isdistributed equally between the daughter cells, which are therefore halfas fluorescent as the parents. As a result, halving of cellularfluorescence intensity marks each successive generation in a populationof proliferating cells and is readily followed by flow cytometry (forfurther details see Hodgkins, P D et al J. Exp. Med. 1996 184:277-281).

Practically, after thawing, PMBC may be washed and stained with CFSEbefore being cultivated (2×10⁵ cells) for 72 hrs with 10 ug/ml ofantigen in culture media (RPMI-1640 supplemented with glutamine, nonessential amino acid, pyruvate and heat inactivated human AB serum).Cells may then then harvested and their phenotype characterised usingsurface staining to identify memory CD8 and CD4+ T-Cells. Subsequently,flow cytometry analysis can be used to indicate the extent of lymphocyteproliferation in response to each antigen (proportion of cells withdecreased CFSE intensity upon in vitro stimulation).

Cytokine Production

IFN-γ production (or the production of other cytokines such as e.g. IL2,TNF-alpha, IL5, IL12 etc) may be measured using an enzyme-linkedimmunosorbent assay (ELISA). ELISA plates may be coated with a mousemonoclonal antibody directed to human IFN-γ (PharMingen, San Diego,Calif.) in PBS for four hours at room temperature. Wells are thenblocked with PBS containing 5% (W/V) non-fat dried milk for 1 hour atroom temperature. The plates are then washed, for example, six times inPBS/0.2% TWEEN™-20 and samples diluted 1:2 in culture medium in theELISA plates are incubated overnight at room temperature. The plates areagain washed and a polyclonal rabbit anti-human IFN-γ serum, forexample, diluted 1:3000 in PBS/10% normal goat serum may be added toeach well. The plates are then incubated for two hours at roomtemperature, washed and horseradish peroxidase-coupled anti-rabbit IgG(Sigma Chemical So., St. Louis, Mo.) may be added, for example, at a1:2000 dilution in PBS/5% non-fat dried milk. After a further two hourincubation at room temperature, the plates are washed and TMB substrateadded. The reaction may be stopped after 20 min with 1 N sulfuric acid.Optical density can then be determined at 450 nm using 570 nm as areference wavelength. Typically, fractions that result in bothreplicates giving an OD two fold greater than the mean OD from cellscultured in medium alone may be considered positive.

Example 4 Immunogenicity in CB6F1 Mice

The immunogenicity of the antigen was evaluated in CB6F1 mice (firstgeneration cross of BALB/c and C57BL/6 mice).

CB6F1 mice were immunised intramuscularly three times (on day 0, day 14and day 28) with 0.5 ug or 2 ug of protein antigen in combination withthe Adjuvant System AS01E (a liposomal adjuvant formulation comprising3D-MPL and QS21).

The experimental design was as follows:

Group Day 0 Day 14 Day 28 1 2 ug Rv2386c/ 2 ug Rv2386c/ 2 ugRv2386c/AS01E AS01E AS01E 2 0.5 ug Rv2386c/ 0.5 ug Rv2386c/ 0.5 ugRv2386c/AS01E AS01E AS01E

A total of 24 mice were used in each protocol group.

Peripheral blood lymphocytes (PBL) were collected and pooled on day 21(i.e. 7 days post second immunisation) and day 35 (i.e. 7 days postthird immunisation) and the antigen-specific CD4 & CD8 T cell responses(as determined by CD4 or CD8 T cells producing IL-2 and/or IFN-gammaand/or TNF-alpha) were measured by flow cytometry after overnight invitro restimulation with pools of 15mer peptides covering the sequencesof interest. The detection of mouse T cells that express IL-2 and/orIFN-gamma and/or TNF-alpha was done by using short-term antigen-drivenin vitro amplification of cytokine expression.

Briefly, PharmLyse solution (BD-Pharmingen) was added to heparinisedmouse peripheral blood in order to lyse the red blood cells. The PBLs(Peripheral Blood Lymphocytes) obtained were washed and then incubatedin the presence of a pool of 15-mer peptides—overlapping by 11 aminoacids—covering the sequence of the antigen of interest and of 1 ug/ml ofantibodies to CD28 and CD49d (BD-Pharmingen). Each 15-mer peptide wasused at a final concentration of 1 ug/ml. Medium controls were alsostimulated with antibodies to CD28 and CD49d.

The cytokine secretion blocking compound brefeldin-A (BD-Pharmingen) wasadded 2 h after the onset of the cultures at 37° C., 5% CO₂ and thecells maintained at 37° C., 5% CO₂ for 4 additional hours followed byovernight incubation at +4° C.

Cells were then harvested and stained with Pacific Blue-coupled anti-CD4(BD—clone RM4-5, BD-Pharmingen) and peridinin chlorophyll A protein(PerCp) cyanin5.5 (Cy5.5)-coupled anti-CD8 alpha (clone 53-6.7,BD-Pharmingen) antibodies.

Cells were then washed, fixed, permeabilised (Cytofix-cytoperm kit,BD-Pharmingen) and stained with allophycocyanin-coupled anti-IFN-gantibodies (clone XMG1.2, BDPharmingen), fluorescein isothiocyanate(FITC)-coupled anti-IL-2 antibodies (clone JES 6-5H4, Beckman Coulter)and phycoerythrin (PE)-coupled anti-TNF alpha antibodies (cloneMP6-XT22, BDPharmingen). After final washes, stained cells were analysedon a LSR II flow cytometer (Beckton-Dickinson). A minimum number of10,000 cells were acquired in the CD8+ subset.

For further background see Walzer T et al Cell Immunol. 2000206(1):16-25 and Maecker H T et al J. Immunol. Methods 2001255(1-2):27-40.

As negative controls, some cells were also cultured overnight in vitroin culture medium (unstimulated). The antigen-specific responses werecalculated by subtracting the average cytokine response produced byunstimulated cells from the average cytokine response produced by thepeptide-stimulated cells.

At each timepoint and for each group, the data was collected from 4pools of 6 mice each. The data below is presented as the % of CD4 or CD8T cells producing IL-2 and/or IFN-gamma and/or TNF-alpha. Eachindividual pool of mice is plotted (triangles) as well as the averagevalue of the group (bar).

FIG. 1 shows that on day 21 (i.e. 7 days post second immunisation),Rv2386c-specific CD4 and CD8 T cell responses are detected in miceimmunised with either dose of Rv2386c/AS01E. The levels ofRv2386c-specific CD4 T cell responses are similar regardless of theimmunising dose of Rv2386. In contrast, mice immunised with 2 ugRv2386c/AS01E displayed higher Rv2386c-specific CD8 T cell responsesthan mice immunised with 0.5 ug Rv2386c/AS01E.

FIG. 2 shows the cytokine profile of CD4 T cell response from theRv2386c peptide pool-stimulated PBL (not medium removed) on day 21 (i.e.7 days post second immunisation).

FIG. 3 shows the cytokine profile of CD8 T cell response from theRv2386c peptide pool-stimulated PBL (not medium removed) on day 21 (i.e.7 days post second immunisation).

FIG. 4 shows that on day 35 (i.e. 7 days post third immunisation),Rv2386c-specific CD4 and CD8 T cell responses are detected in miceimmunised with either dose of Rv2386c/AS01E. The levels ofRv2386c-specific CD4 T cell responses are similar regardless of theimmunising dose of Rv2386. In contrast, mice immunised with 2 ugRv2386c/AS01E displayed higher Rv2386c-specific CD8 T cell responsesthan mice immunised with 0.5 ug Rv2386c/AS01E.

The third immunisation dose increases the CD4 T cell response but notthe CD8 T cell response.

FIG. 5 shows the cytokine profile of CD4 T cell response from theRv2386c peptide pool-stimulated PBL (not medium removed) on day 35 (i.e.7 days post third immunisation).

FIG. 6 shows the cytokine profile of CD8 T cell response from theRv2386c peptide pool-stimulated PBL (not medium removed) on day 35 (i.e.7 days post third immunisation).

Example 5 Immunogenicity in C57BL/6 Mice

The immunogenicity of the antigen was also evaluated in C57BL/6 mice.C57BL/6 mice were immunised intramuscularly three times (on day 0, day14 and day 28) with 1 ug or 4 ug of protein antigen in combination witha the Adjuvant System AS01E (a liposomal adjuvant formulation comprising3D-MPL and QS21).

The experimental design was the following:

Group Day 0 Day 14 Day 28 1 4 ug Rv2386c/ 4 ug Rv2386c/AS01E 4 ugRv2386c/AS01E AS01E 2 1 ug Rv2386c/ 1 ug Rv2386c/AS01E 1 ugRv2386c/AS01E AS01E

Peripheral blood lymphocytes (PBL) were collected and pooled on day 21(i.e. 7 days post second immunisation) and day 35 (i.e. 7 days postthird immunisation) and the antigen-specific CD4 & CD8 T cell responses(as determined by CD4 or CD8 T cells producing IL-2 and/or IFN-gammaand/or TNF-alpha) were measured by flow cytometry after overnight invitro restimulation with pools of 15mer peptides covering the sequencesof interest. The procedure followed was as described previously.

As negative controls, some cells were also cultured overnight in vitroin culture medium (unstimulated). The antigen-specific responses werecalculated by subtracting the average cytokine response produced byunstimulated cells from the average cytokine response produced by thepeptide-stimulated cells.

At each timepoint and for each group, the data was collected from 4pools of 6 mice each. The data below is presented as the % of CD4 or CD8T cells producing IL-2 and/or IFN-gamma and/or TNF-alpha. Eachindividual pool of mice is plotted (triangles) as well as the averagevalue of the group (bar).

FIG. 7 shows that on day 21 (i.e. 7 days post second immunisation),Rv2386c-specific CD4 and CD8 T cell responses are detected in miceimmunised with either dose of Rv2386c/AS01E. The levels ofRv2386c-specific CD4 T cell responses are similar regardless of theimmunising dose of Rv2386c. In contrast, mice immunised with 4 ugRv2386c/AS01E displayed higher Rv2386c-specific CD8 T cell responsesthan mice immunized with 1 ug Rv2386c/AS01E. Due to technicaldifficulties, data for the first pool of cells at the 4 ug dose are notavailable.

FIG. 8 shows the cytokine profile of CD4 T cell response from theRv2386c peptide pool-stimulated PBL (not medium removed) on day 21 (i.e.7 days post second immunisation). Due to technical difficulties, datafor the first pool of cells at the 4 ug dose are not available.

FIG. 9 shows the cytokine profile of CD8 T cell response from theRv2386c peptide pool-stimulated PBL (not medium removed) on day 21 (i.e.7 days post second immunisation). Due to technical difficulties, datafor the first pool of cells at the 4 ug dose are not available.

Immunological data for day 35 were not yet available at the time thisapplication was prepared.

In conclusion it may be noted that the Rv2386c antigen is capable ofeliciting an immune response in both CB6F1 and C57BL/6 mice.Furthermore, the profile of cytokine production indicates that a largeproportion of antigen-specific T-cells express a plurality of Th1associated cytokines (i.e. a polyfunctional T-cell response iselicited). Importantly both CD4 and CD8 antigen-specific T-cells arepresent after immunisation, CD8 cells may be particularly important in alatent TB scenario. Rv2386c may therefore be expected to be ofsubstantial value in the prevention, treatment and diagnosis of latenttuberculosis infection.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

All references referred to in this application, including patents andpatent applications, are incorporated herein by reference to the fullestextent possible as if each individual publication or patent applicationwere specifically and individually indicated to be incorporated byreference.

THROUGHOUT THE SPECIFICATION AND THE CLAIMS WHICH FOLLOW, UNLESS THECONTEXT REQUIRES OTHERWISE, THE WORD ‘COMPRISE’, AND VARIATIONS SUCH AS‘COMPRISES’ AND ‘COMPRISING’, WILL BE UNDERSTOOD TO IMPLY THE INCLUSIONOF A STATED INTEGER, STEP, GROUP OF INTEGERS OR GROUP OF STEPS BUT NOTTO THE EXCLUSION OF ANY OTHER INTEGER, STEP, GROUP OF INTEGERS OR GROUPOF STEPS.

We claim:
 1. A sterile pharmaceutical composition comprising apolypeptide which comprises: (i) an Rv2386c protein comprising an aminoacid sequence selected from the group consisting of SEQ ID NOS: 1 and 6;or (ii) an Rv2386c protein comprising an amino acid sequence at least90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 1 and 6; and a pharmaceutically acceptablecarrier or excipient.
 2. An immunogenic composition comprising apolypeptide which comprises: (i) an Rv2386c protein comprising an aminoacid sequence selected from the group consisting of SEQ ID NOS: 1 and 6;or (ii) an Rv2386c protein comprising an amino acid sequence at least90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 1 and 6; and a non-specific immune responseenhancer.
 3. The composition according to claim 1, wherein thepolypeptide comprises an amino acid sequence at least 90% identical toSEQ ID NO:
 1. 4. The immunogenic composition according to claim 2,wherein the polypeptide comprises an amino acid sequence at least 90%identical to SEQ ID NO:1.
 5. The composition according to claim 1,wherein the polypeptide comprises an amino acid sequence at least 90%identical to SEQ ID NO:
 6. 6. The immunogenic composition according toclaim 2, wherein the polypeptide comprises an amino acid sequence atleast 90% identical to SEQ ID NO:6.